Mglu receptors antagonists for treating disorders associated with mglu receptors including addiction and depression

ABSTRACT

Methods are provided for treating disorders associated with mGlu receptors by simultaneously inhibiting at least two mGIuRs belonging to at least two different groups. In one embodiment, there are provided methods for treating a disorder associated with MGlu receptors 2, 3, and 5, including administering to a subject in need thereof an effective amount of at least one antagonist which modulates mGIuR2, mGIuR3, and mGIuRS. The disorders treated by the method include, for example, nicotine addiction, cocaine addiction, and depression.

FIELD OF THE INVENTION

The present invention relates generally to methods for treatingdisorders associated with metabotropic glutamate receptors, and morespecifically to methods for treating disorders associated withmetabotropic glutamate receptors 2, 3, and 5.

BACKGROUND INFORMATION

Glutamate receptors play a role in numerous neurological,neurodegenerative, psychiatric, and psychological disorders, and avariety of mammalian disease states are associated with aberrantactivity of these receptors. Glutamate receptors have been classified aseither “ionotropic” or “metabotropic”. Ionotropic receptors are directlycoupled to the opening of cation channels in the cell membranes of theneuron. Metabotropic receptors belong to the family of G-protein-coupledreceptors and are coupled to systems that lead to enhancedphosphoinositide hydrolysis, activation of phospholipase D, increases ordecreases in cAMP formation, and changes in ion channel function.

Metabotropic glutamate receptors (mGluRs) are divided into three groupsbased on amino acid sequence homology, transduction mechanism andbinding selectivity: Group I, Group II and Group III. Group I includesmetabotropic glutamate receptors 1 and 5 (mGluR1 and mGluR5), Group IIincludes metabotropic glutamate receptors 2 and 3 (mGluR2 and mGluR3),and Group III includes metabotropic glutamate receptors 4, 6, 7, and 8(mGluR4, mGluR6, mGluR7 and mGluR8). Each mGluR type may be found inseveral subtypes. For example, subtypes of mGluR1 include mGluR1a,mGluR1b and mGluR1c.

It is only recently that researchers have begun to elucidatephysiological roles for each mGluR group. For example, Group IImetabotropic glutamate receptors (mGluII), including mGlu2 and mGlu3receptors, are inhibitory autoreceptors located primarily onglutamatergic afferents throughout the mammalian brain where theydecrease excitatory glutamate transmission (Cartmell and Schoepp, JNeurochem 75:889-907, 2000). GABA_(B) receptors, which share closestructural and functional homology to mGluII receptors (Schoepp, J.Pharmacol. Exp. Ther., 299:12-20, 2001), also negatively regulateglutamate transmission. Recently, activation of mGluII and GABA_(B)receptors was shown to decrease excitatory glutamate transmission in theventral tegmental area (VTA) and nucleus accumbens (NAcc) (Bonci et al.,Eur. J. Neurosci., 9:2359-2369, 1997; Xi et al., J. Pharmacol. Exp.Ther., 300:162-171, 2002; Erhardt et al., Naunyn Schmiedebergs Arch.Pharmacol., 365:173-180, 2002), suggesting that these receptors mayregulate the activity of the brain's reward circuitry. Accordingly,LY314582 and CGP44532, agonists at mGluII and GABA_(B) receptorsrespectively, were shown to elevate intracranial self-stimulation (ICSS)reward thresholds in drug-naïve rats (Macey et al., Neuropharmacology,40:676-685, 2001; Harrison et al., Psychopharmacology, 160:56-66, 2002),demonstrating that mGluII and GABA_(B) receptors negatively regulatebrain reward function.

Moreover, there is accumulating evidence that the function of mGluII andGABA_(B) receptors increases during the development of drug dependence.For example, prolonged morphine, cocaine or amphetamine treatmentincreased inhibitory regulation of glutamate transmission by mGluII andGABA_(B) receptors located in the VTA and NAcc (Manzoni and Williams, J.Neurosci., 19:6629-6636,1999; Xi et al., Soc. Neurosci., Abstr 27: 2596,2001; Giorgetti et al., Neuroscience, 109:585-595, 2002).

Attempts at elucidating the physiological roles of Group I mGluRssuggest that activation of these receptors elicits neuronal excitation.Evidence indicates that this excitation is due to direct activation ofpostsynaptic mGluRs, but it also has been suggested that activation ofpresynaptic mGluRs occurs, resulting in increased neurotransmitterrelease (Baskys, Trends Pharmacol. Sci. 15:92,1992, Schoepp, Neurochem.Int. 24:439, 1994, Pin et al., Neuropharmacology 34:1, 1995.) Thus, ithas been proposed that antagonists for the Group I mGluRs may be usefulin treating neurological disorders such as senile dementia, Parkinson'sdisease, Alzheimer's disease, Huntington's Chorea, pain, epilepsy, andhead trauma.

However, less is known about the potential therapeutic benefits that maybe realized as a result of simultaneous antagonism of mGluRs belongingto different groups. Furthermore, little is known about whetherantagonists of mGluRs are useful for treating disorders such assubstance abuse and depression. The invention addresses these issues andfurther provides related advantages.

SUMMARY OF THE INVENTION

The present invention provides methods for treating disorders associatedwith metabotropic glutamate receptors (mGluRs) by simultaneouslyinhibiting at least two mGluRs belonging to at least two differentgroups. In one embodiment, there are provided methods for treating ametabotropic glutamate disorder, including administering to a subject inneed thereof an effective amount of at least one antagonist whichmodulates mGluR2, mGluR3, and mGluR5.

In another embodiment, the present invention provides methods fortreating a metabotropic glutamate disorder including administering to asubject in need thereof an effective amount of at least one antagonistwhich modulates mGluR2 and mGluR5, thereby treating the disorder.

In still another embodiment, the present invention provides methods fortreating a metabotropic glutamate disorder, including administering to asubject in need thereof an effective amount of at least one antagonistwhich modulates mGluR3 and mGluR5, thereby treating the disorder.

In still another embodiment, the present invention provides methods fortreating substance abuse. In one aspect, a method according to thisembodiment includes administering to a subject in need thereof aneffective amount of at least one antagonist which modulates mGluR2,mGluR3, and mGluR5, wherein the effective amount is sufficient todiminish, inhibit or eliminate desire for said substance in saidsubject.

In still another embodiment, the present invention provides methods fortreating depression, including administering to a subject in needthereof an effective amount of at least one antagonist which modulatesmGluR2, mGluR3 and/or mGluR5, thereby treating the depression. Thedepression can be either drug-induced or non-drug induced depression.

In another embodiment, the present invention provides a method ofscreening for an agent that improves the ability of an mGluR2, mGluR3,and/or mGluR5 antagonist to at least partially normalize a deficit inbrain reward function reflected in intracranial self-stimulation (ICSS)threshold of a non-human mammalian subject. The method includes:

-   -   a) affecting the ICSS threshold of the subject;    -   b) administering to the subject, a sufficient amount of the        known inhibitor to at least partially normalize the ICSS        threshold when administered alone or in combination with another        inhibitor, wherein the known inhibitor is an antagonist of at        least one of mGluR2, mGluR3, and mGluR5;    -   b) administering to the non-human mammalian subject, an        effective amount of a test agent, wherein the test agent is a        known or suspected antagonist of at least one of mGluR2, mGluR3,        and mGluR5; and    -   c) determining whether the test agent improves the ability of        the known inhibitor to at least partially normalize the ICSS        threshold, thereby identifying an agent that improves the        ability of the known inhibitor to at least partially normalize        ICSS threshold.

In one aspect, the method includes administering an mGluR2 and/or mGluR3inhibitor, such as LY341495, simultaneously with a test agent that isknown or suspected to be an antagonist of mGluR5. In another aspect, themethod includes administering an mGluR5 inhibitor, such as MPEP,simultaneously with a test agent that is know or suspected to be anantagonist of mGluR2 and/or mGluR3.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of coronal sections from the ratbrain showing histological reconstruction of the injection sites in theventral tegmental area (5.30-6.72 mm posterior to bregma, according tothe atlas of Paxinos and Watson, 1986). Black circles indicate locationsof injection tips located inside the VTA and included in statisticalanalysis. Data from rats with injection sites located outside the VTAwere removed from the analyses.

FIGS. 2A-2B illustrate the effects of LY314582 on ICSS thresholds andresponse latencies in nicotine-treated and control rats. FIG. 2A, Dataare expressed as mean (±SEM) percentage change from baseline threshold.FIG. 2B, Data are expressed as mean (±SEM) percentage change frombaseline response latency. **P<0.01, different from nicotine-treatedrats after vehicle injection. ##P<0.01, different from control ratsafter injection with same dose of LY314582.

FIGS. 3A-3B illustrate the effects of intra-ventral tegmental areaLY314582 on ICSS thresholds and response latencies in nicotine-treatedand control rats. FIG. 3A, Data are expressed as mean (±SEM) percentagechange from baseline threshold. FIG. 3B, Data are expressed as mean(±SEM) percentage change from baseline response latency. ***P<0.001,different from nicotine-treated rats after vehicle injection. ##P<0.01,#p<0.05, different from control rats after injection with same dose ofLY314582.

FIGS. 4A-4B illustrate the effects of LY341495 on the elevations in ICSSthresholds in rats undergoing spontaneous nicotine withdrawal. FIG. 4A,Data are expressed as mean (±SEM) percentage change from baselinethreshold in rats undergoing nicotine withdrawal. FIG. 4B, Data areexpressed as mean (±SEM) percentage change from baseline threshold incontrol rats. ICSS thresholds and response latencies were tested 12, 18,24, 36, 48 and 72 h after surgical removal of osmotic mini-pumpsdelivering nicotine (FIG. 3A) or vehicle (FIG. 3B). Rats received asingle injection of LY341495 (1 mg/kg) or vehicle 30 min before the 18 htime-point (indicated by black arrow). ***P<0.001, different from ratsundergoing nicotine withdrawal treated with vehicle 30 min before the 18h time-point.

FIG. 5 illustrates the effects of NBQX on ICSS thresholds and responselatencies in nicotine-treated and control rats. A, Data are expressed asmean (±SEM) percentage change from baseline threshold. B, Data areexpressed as mean (±SEM) percentage change from baseline responselatency. *P<0.05, **p<0.01, different from nicotine-treated rats aftervehicle injection. #P<0.05, ##p<0.01, different from control rats afterinjection with same dose of NBQX.

FIG. 6 illustrates the effects of MPEP administration on nicotine- andfood-maintained responding in the rat. The data are expressed as percentof baseline responding (mean±SEM). Asterisks indicate significantdifferences from control conditions for each reinforcer (*p<0.05,**p<0.01).

FIGS. 7A-7D illustrate nicotine dose-response curves obtained afterpretreatment with different doses of MPEP (mean±SEM). The graphs in thisfigure depicts the nicotine dose-response curves obtained afterpretreatment with 0 (FIG. 7A, 5 (FIG. 7B), 10 (FIG. 7C) and 20 (FIG. 7D)mg/kg MPEP. The filled circles are results from saline controls (thesame data repeated in all four panels) and open squares are results fromMPEP treated subjects. The asterisk (*) indicates significant (p<0.05)differences in R-criterion value for different available nicotine dosescompared to saline. The pound signs (#) indicate significant differencesfrom self-administration of the 0.048 μg/inf nicotine dose for each MPEPdose (5, 10 and 20 mg/kg) compared to saline pretreatment (p<0.05).

FIGS. 8A-8B illustrate the effect of MPEP administration on intravenousself-administration of 0.048 μg/inf nicotine in DBA/2J mice (mean±SEM).Panel A (left panel) depicts the effects of MPEP pretreatment onself-administration of this reliably self-administered nicotine dose(0.048 Tg/inf nicotine). Panel B (right panel) shows total self-injectednicotine dose when 0.048 μg/inf nicotine was available afterpretreatment with MPEP (0, 5, 10 and 20 mg/kg). Asterisks (*) indicatesignificant differences after pretreatment with each MPEP dose (5, 10and 20 mg/kg) compared to saline pretreatment (p<0.05).

FIGS. 9A-9B illustrate the effects of MPEP administration on cocaineresponding in Short Access (ShA) and Long Access (LgA) rats. FIG. 9Ashows development of escalation In cocaine Intake In the LgA rats; dataare express as raw values. FIG. 9B shows the effects of MPEP In ShA andLgA rats as percent of baseline responding (mean±SEM). Panel C Indicatesthe effects of MPEP on cocaine self-administration (data expressed aspercent of baseline responding (mean+SEM) with ShA and LgA ratscombined). Asterisks indicate significant differences from controlconditions for each reinforcer (*P<0.05, **P<0.01).

FIG. 10 illustrates the effects of MPEP on cocaine-, nicotine- andfood-maintained responding under a progressive-ratio schedule ofreinforcement. Data are presented as the mean (±SEM) number ofinfusions/food pellets earned after MPEP pretreatment (left ordinalaxis). The right ordinal axis shows the corresponding final ratios(i.e., break-points) reached.

FIG. 11 illustrates the effects of MPEP administration on the magnitudeof cocaine-induced lowering of ICSS reward thresholds (a) and effects onresponse latencies in the same procedure (b). The data are expressed aspercent of baseline (mean±SEM). *P<0.05, ***p<0.001 from control;#p<0.05 compared with rats similarly treated with the same dose of MPEP,but did not receive cocaine.

FIG. 12 illustrates the effects of MPEP administration on the durationof cocaine-induced lowering of ICSS reward thresholds (a) and theeffects on response latencies (b). The data are expressed as percent ofbaseline (mean±SEM). ***p<0.001 from control.

FIG. 13 illustrates the effects of LY341495 (0.1-5 mg/kg) administrationon nicotine responding in rats. The data are expressed as percent ofbaseline responding (mean±SEM).

FIG. 14 illustrates the effects of LY341495 (0.5 mg/kg) alone, or incombination with a dose of MPEP (1 mg/kg) previously shown (see FIG. 13above) to have no effect by itself on cocaine consumption, on nicotineresponding in rats. The data are expressed as percent of baselineresponding (mean±SEM).

FIG. 15 illustrates the effects of LY341495 (1 mg/kg) combined with MPEP(1 mg/kg) on nicotine responding in rats. The data are expressed aspercent of baseline responding (mean±SEM).

FIG. 16 illustrates the effects of LY341495 (1 mg/kg) and of the drugcombination LY341495 and MPEP (1 mg/kg and 9 mg/kg, respectively) onbreak-points (i.e., highest fixed ratio attained depicted on the righty-axis) and number of nicotine injections earned (depicted on the lefty-axis) under a progressive ratio of reinforcement that reflects themotivation for drug-taking behavior. MPEP potentiated the break-pointdecreases induced by LY341495). Data are presented as the mean (±SEM)number of infusions earned after the drug treatments (left ordinalaxis). The right ordinal axis shows the corresponding final ratios(i.e., break-points) reached.

FIGS. 17A-17B illustrate the effects of the selective serotonin reuptakeinhibitor paroxetine on brain reward thresholds (A) and responselatencies (B) (mean±SEM).

FIGS. 18A-18B illustrate the effects of the serotonin-1A receptorantagonist, p-MPPI, and combinations of p-MPPI+paroxetine on brainstimulation reward thresholds (A) and response latencies (B) (mean±SEM).Asterisks (*) denote a statistically significant linear trend (p<0.05).

FIGS. 19A-C illustrate the effects of chronic amphetamine administrationon reward thresholds (A), response latencies (B) and body weight (C)(mean±SEM). Asterisks (*) denote statistically significant differencesfrom saline-exposed control animals (p<0.05). Hashes (#) denotestatistically differences from drug day 1 (before any drugadministration; baseline day) within each drug group (p<0.05). Crosses(+) denote statistically significant differences from drug day 2 withineach drug group. (%) denotes statistically differences from drug day 3within each drug group (p<0.05). Filled squares represent data fromasaline treated rats. Open circles represent data from amphetaminetreated rats.

FIGS. 20A-F illustrate the effects of serotonergic treatments onamphetamine withdrawal-induced reward deficits. FIGS. 20A-20C depictthresholds of saline-exposed subjects treated acutely with the varioustreatments; for clarity the same saline-exposed vehicle-treated controlgroup is presented in each panel. FIGS. 20D-20F depict thresholds ofamphetamine-exposed subjects; the same amphetamine-exposedvehicle-treated group is presented in each figure. The arrow indicatesthe time-point at which the acute drug treatment was administered.Asterisks (*) denote statistically significant differences between thedrug combination- and saline-treated groups (p<0.05) (triangles). Hashes(#) denote significant differences from the saline-exposedvehicle-treated rats (p<0.05) (squares).

FIG. 21 illustrates the effects of amphetamine withdrawal andserotonergic treatments on response latencies (mean±SEM). The arrowindicates the time-point at which the acute drug treatment wasadministered. Asterisks (*) denote statistically significant differencesfrom the vehicle control group (p<0.05). Squares represent data pointsfrom vehicle-treated rats (n=22). Triangles represent data from p-MPPItreated rats (3 mg/kg; n=22). Filled circles represent data fromparoxetine treated rats (1.25 mg/kg; n=21). Open circles represent datafrom rats treated with paroxetine and p-MPPI (n=24).

FIGS. 22A-22D illustrate the effects of amphetamine withdrawal andserotonergic treatments on body weight (mean±SEM). The arrow indicatesthe time-point at which the acute drug treatment was administered.Asterisks (*) denote statistically significant differences between thesaline- and amphetamine-exposed groups (p<0.05). Hashes (#) denotestatistically significant differences from the 12 hr time point (afterchronic treatment and before the acute treatment) within each drug group(p<0.05). Closed squares represent saline-treated groups and opensquares represent amphetamine-treated groups. For saline-treated groups,FIG. 22A (Vehicle treatment), n=1 1; FIG. 22B (p-MPPI (3 mg/kg)treatment), n=10, FIG. 22C (Paroxetine (1.25 mg/kg) treatment), n=10;and FIG. 22D [p-MPPI (3 mg/kg) and paroxetine (1.25 mg/kg) treatment],n=12.

FIG. 23 illustrates the effects of acute treatment with theantidepressant bupropion on brain reward thresholds. Bupropion at alldoses (n=8) tested and in a dose-dependent fashion up to 40 mg/kgresulted in a reduction in brain reward thresholds when compared withvehicle-treated animals, which is indicative of an enhancement of brainreward function. All bars represent mean values with vertical linesindicating 1 SEM. * indicates groups that differed significantly fromvehicle-treated animals; P<0.05. Open bar is Vehicle; horizontal linebar is 10 mg/kg, upper left to lower right lined bar is 20 mg/kg; lowerleft to upper right lined bar is 30 mg/kg; cross-hatched bar is 40mg/kg; and vertical line bar is 60 mg/kg.

FIG. 24 illustrates the effects of the antidepressant bupropion onnicotine (0.25 mg/kg)-induced enhancement of brain reward function.Bupropion administration (10 and 20 mg/kg) resulted in a lowering ofbrain reward thresholds (n=1 0). Nicotine treatment resulted in asimilar reduction in reward thresholds. This rewarding effect ofnicotine was completely countered by bupropion (5 mg/kg) pretreatment,which was without effect on reward thresholds when given alone. All barsrepresent mean values with vertical lines indicating 1 SEM. * indicatesthresholds that differed significantly from the relevant vehiclecondition; P<0.05. # indicates groups that differed significantly fromrelevant saline co-treated control; P<0.05. Left group of bars are fromsaline-treated rats. Right group of bars are from nicotine (0.25 mg/kg)treated rats. Open bar is Vehicle; upper left to lower right lined baris 5 mg/kg; lower left to upper right lined bar is 10 mg/kg;cross-hatched bar is 20 mg/kg.

FIG. 25 illustrates the effects of continuous infusion of nicotine (3.16mg/kg free base per day for 7 days) on brain reward thresholds. Nicotineadministration (n=38) resulted in a time-dependent lowering of brainreward thresholds compared to animals prepared with saline pumps (n=38).The peak threshold lowering effect was on day 3. Thresholds returned tobaseline levels by day 7. All data points represent mean values withvertical lines indicating 1 SEM. Inset: The area under the curveanalysis clearly shows that animals treated with nicotine hadsignificantly lower thresholds over the duration of the 7 days oftreatment. All bars represent mean values with vertical lines indicating1 SEM. * indicates thresholds that differed significantly from thesaline-treated animals; P<0.05. Open bar and squares representsaline-treated rats. Filled bar and squares represent nicotine-treatedrats.

FIGS. 26A-26B illustrate the effects of the antidepressant bupropion onbrain reward thresholds following withdrawal from chronic nicotine (3.16mg/kg per day for 7 days) or saline administration. As illustrated inFIG. 26A, in saline pretreated animals, bupropion treatment resulted ina lowering of brain reward thresholds at all doses (10 mg/kg, n=8; 20mg/kg, n=8; 40 mg/kg, n=10) compared with vehicle-treated subjects(n=12). This threshold lowering was short lived as all animals returnedto baseline thresholds at the next testing point that was 6 h later. Allnicotine-pretreated animals exhibited elevated thresholds 12 h followingwithdrawal. Bupropion treatment before the 18-h time point resulted in alowering of brain reward thresholds at all doses tested (10 mg/kg, n=9;20 mg/kg, n=9; 40 mg/kg, n=9) compared with vehicle-treated subjectsthat were exposed to nicotine (n=1 1). This reversal of thresholdelevations was significantly prolonged in animals treated with thehigher bupropion dose (40 mg/kg) that were previously treated withnicotine; these animals exhibited thresholds significantly lower thansaline-treated nicotine withdrawing subjects at the 24-h withdrawal timepoint (6.5 h post-bupropion administration). All data points representmean values with vertical lines indicating 1 SEM. * indicates groupsthat differed significantly from corresponding vehicle treated animals;P<0.05. # indicates groups that differed significantly from relevantchronic saline-pretreated controls; P<0.05. Open squares indicatevehicle; Up arrows indicate 10 mg/kg bupropion; Down arrows indicate 20mg/kg bupropion; Circles represent 40 mg/kg bupropion. FIG. 26Billustrates that the area under the curve analysis also shows thatanimals pretreated with nicotine had elevated brain reward thresholdsafter termination of nicotine administration. (i.e., withdrawal).Furthermore, this analysis clearly demonstrates that acute treatmentwith bupropion (40 mg/kg) reversed this elevation in thresholds. Allbars represent mean values with vertical lines indicating 1 SEM. *indicates groups that differed significantly from correspondingvehicle-treated animals; P<0.05. # indicates groups that differedsignificantly from relevant chronic saline pretreated controls; P<0.05.Open bar is Vehicle; upper left to lower right lined bar is 10 mg/kg;lower left to upper right lined bar is 20 mg/kg; cross-hatched bar is 40mg/kg.

FIGS. 27A-B illustrate the effects of the antidepressant bupropion onsomatic signs 12 h following withdrawal from chronic nicotine treatment(3.16 mg/kg per day for 6.75 days). As illustrated in FIG. 27A, sixhours following withdrawal from chronic nicotine administration, therewas a significant increase in the amount of total somatic signs ofabstinence. Bupropion treatment 30 min prior to the 12-h withdrawal timepoint resulted in a reversal of the expression of somatic signs (5mg/kg, n=8; 10 mg/kg, n=7; 20 mg/kg, n=7; 40 mg/kg, n=8) compared withvehicle treatment (n=6). FIG. 27B illustrates the effects of bupropionon the individual clusters of signs of withdrawal 12 h followingminipump removal. All bars represent mean values with vertical linesindicating 1 SEM. * indicates groups that differed significantly fromvehicle-treated animals; P<0.05. # indicates groups that differedsignificantly from baseline (6 h following initiation of withdrawal)level; P<0.05. Open bars represent Vehicle; horizontal bars represent 5mg/kg bupropion; upper left to lower right lined bars represent 10mg/kg; lower left to upper right lined bars represent 20 mg/kg;cross-hatched bars represent 40 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the following findings:

a) Blockade of mGlu2 and mGlu3 receptors by an antagonist of mGluR2 andmGluR3 (also referred to herein as an “mGlu2/3 receptor antagonist”),for example by administration of the mGlu2/3 receptor antagonistLY341495, attenuates the depression-like aspects of nicotine withdrawalin rats;

b) Treatment with an mGlu5 receptor antagonist, such as MPEP, decreasescocaine and nicotine consumption in rats and mice.

c) Treatment with an antagonist of mGlu2 and mGlu3 receptors, such asLY341495, decreases nicotine consumption in rats;

d) Co-administration of a dose of an mGlu5 receptor antagonist, such asMPEP at 1 mg/kg, that has no effects on cocaine or nicotineself-administration potentiates the inhibitory effects of an mGlu2/3receptor antagonist, such as LY341495 (0.5 mg/kg or 1 mg/kg), onnicotine self-administration; and

e) The combination of an mGlu5 receptor antagonist at a concentrationthat decreases either cocaine or nicotine self-administration whenadministered alone, e.g., 9 mg/kg MPEP, when combined with an mGlu2/3receptor antagonist at a concentration that decreases nicotineself-administration when administered alone, such as 1 mg/kg LY341495,is more effective at decreasing nicotine self-administration than anyone drug alone.

Based on these findings, the present invention provides a method fortreating a metabotropic glutamate disorder that includes administeringto a subject in need thereof, an effective amount of at least oneantagonist which modulates mGluR2, mGluR3, and mGluR5, thereby treatingthe disorder. In another embodiment, methods are provided for treating ametabotropic glutamate disorder including administering to a subject inneed thereof an effective amount of at least one antagonist whichmodulates mGluR2 and mGluR5, thereby treating the disorder. In stillanother embodiment, methods are provided for treating a metabotropicglutamate disorder including administering to a subject in need thereofan effective amount of at least one antagonist which modulates mGluR3and mGluR5, thereby treating the disorder. In still another embodiment,methods are provided for treating a metabotropic glutamate disorderincluding administering to a subject in need thereof an effective amountof at least one antagonist which modulates mGluR2 and mGluR3 and oneantagonist that modulates mGluR5, thereby treating the disorder. Instill another embodiment, methods are provided for treating ametabotropic glutamate disorder including administering to a subject inneed thereof an effective amount of at least one antagonist whichmodulates mGluR2 and mGluR3, thereby treating the disorder. Because ofthe known similarities of mGluR1 and mGluR5 (both belonging to Group I),an antagonist of mGluR1 can be used in place of an mGluR5 antagonist inany of the methods of the present invention.

In certain embodiments of the invention, simultaneous antagonism ofmetabotropic glutamate receptors 2, 3 and 5, is accomplished byadministering to a subject in need thereof, one antagonist which acts asan inhibitor of all three receptors. Alternatively, a combination ofantagonists can be used to achieve inhibition of receptors 2, 3, and 5.For example, administration of an antagonist of mGluR2 and mGluR3 incombination with an antagaonist of mGluR 5 is used in certain aspects ofthe invention, to achieve inhibition of mGluRs 2, 3, and 5. For example,the mGluR2 and mGluR3 antagonist LY341495, in combination with themGluR5 antagonist MPEP, can be administered to a subject.

Furthermore, the present invention provides

-   a combination comprising (a) at least one active ingredient selected    from a metabotropic glutamate receptor 2 antagonist and a    metabotropic glutamate receptor 3 antagonist, and (b) at least one    metabotropic glutamate receptor 5 antagonist, in which the active    ingredients are present in each case in free form or in the form of    a pharmaceutically acceptable salt, and optionally at least one    pharmaceutically acceptable carrier; for simultaneous, separate or    sequential use, especially in the treatment of an addicitve disorder    or depression;-   a combination comprising (a) at least one active ingredient which    exhibits antagonistic activity against the metabotropic glutamate    receptor 2 and the metabotropic glutamate receptor 3, and (b) at    least one metabotropic glutamate receptor 5 antagonist, in which the    active ingredients are present in each case in free form or in the    form of a pharmaceutically acceptable salt, and optionally at least    one pharmaceutically acceptable carrier; for simultaneous, separate    or sequential use, especially in the treatment of an addicitve    disorder or depression;-   a combination comprising (a) at least one metabotropic glutamate    receptor 2 antagonist, and (b) at least one active ingredient which    exhibits antagonistic activity against the metabotropic glutamate    receptor 3 and the metabotropic glutamate receptor 5, in which the    active ingredients are present in each case in free form or in the    form of a pharmaceutically acceptable salt, and optionally at least    one pharmaceutically acceptable carrier; for simultaneous, separate    or sequential use, especially in the treatment of an addicitve    disorder or depression; and-   a combination comprising (a) at least one metabotropic glutamate    receptor 3 antagonist, and (b) at least one active ingredient which    exhibits antagonistic activity against the metabotropic glutamate    receptor 2 and the metabotropic glutamate receptor 5, in which the    active ingredients are present in each case in free form or in the    form of a pharmaceutically acceptable salt, and optionally at least    one pharmaceutically acceptable carrier; for simultaneous, separate    or sequential use, especially in the treatment of an addicitve    disorder or depression.

The combinations mentioned above can be applied in the form of acombined preparation or a pharmaceutical composition.

Additionally, the present invention relates to the following aspects:

a method of treating a warm-blooded animal having an addicitve disorderor depression comprising administering to the animal a combinationaccording defined above in a quantity which is jointly therapeuticallyeffective against an addicitve disorder or depression and in which thecompounds can also be present in the form of their pharmaceuticallyacceptable salts;

a pharmaceutical composition comprising a quantity, which is jointlytherapeutically effective against an addicitve disorder or depression,of a pharmaceutical combination as defined above and at least onepharmaceutically acceptable carrier;

the use of a combination as defined above for the preparation of amedicament for the treatment of an addicitve disorder or depression; and

a commercial package comprising a combination defined above togetherwith instructions for simultaneous, separate or sequential use thereofin the treatment of an addicitve disorder or depression.

The term “a combined preparation”, as used herein defines especially a“kit of parts” in the sense that the first and second active ingredientas defined above can be dosed independently or by use of different fixedcombinations with distinguished amounts of the ingredients, i.e.,simultaneously or at different time points. The parts of the kit ofparts can then, e.g., be administered simultaneously or chronologicallystaggered, that is at different time points and with equal or differenttime intervals for any part of the kit of parts. Very preferably, thetime intervals are chosen such that the effect on the treated disease inthe combined use of the parts is larger than the effect which would beobtained by use of only any one of the active ingredients. The ratio ofthe total amounts of the active ingredient 1 to the active ingredient 2to be administered in the combined preparation can be varied, e.g., inorder to cope with the needs of a patient sub-population to be treatedor the needs of the single patient which different needs can be due toage, sex, body weight, etc. of the patients. Preferably, there is atleast one beneficial effect, e.g., a mutual enhancing of the effect ofthe first and second active ingredient, in particular a synergism, e.g.a more than additive effect, additional advantageous effects, less sideeffects, a combined therapeutic effect in a non-effective dosage of oneor both of the first and second active ingredient, and especially astrong synergism the first and second active ingredient.

The pharmacological activity of a combination as defined above may, forexample, be evidenced in preclinical studies known as such, e.g. inanalogy to those described in the Examples.

The pharmacological activity of a combination as defined above may, forexample, also be demonstrated in a clinical study. Such clinical studiesare preferably randomized, double-blind, clinical studies in patientswith addictive disorders or depression. Such studies demonstrate, inparticular, the synergism of the active ingredients of the combinationas defined above. The beneficial effects on addictive disorders ordepression can be determined directly through the results of thesestudies or by changes in the study design which are known as such to aperson skilled in the art.

Antagonists and agonists of mGlu receptors are known in the art.Examples of some known antagonists and agonists are provided in theTable below and in the following paragraphs. Agonists includenon-selective agonists: 1S,3 R-ACPD; 1S,3 S-ACPD; L-CCG-I. Antagonistsinclude broad-spectrum, non-selective antagonists such as (S)-MCPG. Itwill be understood that based on the teachings of the present invention,virtually any mGluR 1, 2, 3, and/or 5 antagonist can be used with themethods of the present invention. However, preferably for the presentinvention, antagonists that are selective for mGluR1, R2, R3 and/or R5are used. Antagonists of mGluR2 are known in the art and include, forexample, the compounds disclosed in U.S. Pat. No. 6,407,094 (Adam etal., (2002), incorporated in its entirety herein by reference) and1-[(Z)-2-Cycloheptyloxy-2-(2,6-dichloro-phenyl)-vinyl]-1H-[1,2,4]triazole (Kolczewski et al. Bioorg. Med. Chem. Lett. 1999, 9,2173-2176). Antagonists of both mGluR2 and mGluR3 are known in the artand include, for example, LY341495 (Kingston et al. Neuropharm. 1998,37, 1-12)., LY366457 (O'Neill M. F., et al., Neuropharmacology,45(5):565-74 (2003)), (2S)-alpha-ethylglutamic acid (EGLU) (See e.g.,Neto, F. L., et al., Neurosci. Lett. 15;296(1):25-8 (2000)), and(2S,4S)-amino-4-(2,2-diphenylethyl)pentanedioic acid (Escribano, A., etal., Bioorg. Med. Chem. Lett., 7;8(7):765-70 (1998)). Antagonists ofmGluR5 are known in the art and include, for example, MPEP(2-methyl-6-(phenylethynyl)pyridine) and MTEP(3-(2-Methyl-thiazol-4-ylethynyl)-pyridine (Cosford, N. D., et al., J.Med. Chem., 46(2):204-6 (2003)). Antagonists of mGluR1 include, forexample, 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA), (Moroni,F., Neuropharmacology, 42(6):741-51 (2002)). Antagonists of mGluR1 andmGluR5 (Group I antagonists), include, for example,1-aminoindan-1,5-dicarboxylic acid (AIDA) (See e.g., Renaud, J., et al.,Epilepsia, 43(11):1306-17 (2002)),(3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on(BAY 36-7620) (See e.g., De Vry, J., et al., Eur. J. Pharmacol.5;428(2):203-14 (2001)), and the cyclobutylglycine(+/−)-2-amino-2-(3-cis andtrans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053)(Chen, U., et al., Neuroscience, 95(3):787-93 (2000)). Other mGluRantagonists that can be used in the present invention include thosedisclosed in WO99/08678, incorporated in its entirety herein byreference.

The structure of the active ingredients identified by code nos., genericor trade names may be taken from the actual edition of the standardcompendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications). The corresponding contentthereof is hereby incorporated by reference. Any person skilled in theart is fully enabled to identify the active ingredients and, based onthese references, likewise enabled to manufacture and test thepharmaceutical indications and properties in standard test models, bothin vitro and in vivo. TABLE Table of mGluR Agonists and AntagonistsReceptor Group Subtype Receptor Agonist Receptor Antagonist Group ImGlu1 (S)-3,5-DHPG LY 367385 Quisqualate (S)-4-CPG CPCCOEt mGlu5(S)-3,5-DHPG MPEP (RS)-CHPG (S)-4-CPG Z-CBQA MTEP Quisqualate Group IImGlu2 LY354740 LY341495 LY379268 ADED (2R,4R)-APDC EGlu DCG-IV MSOPmGlu3 LY354740 LY341495 LY379268 ADED (2R,4R)-APDC DCG-IV Group IIImGlu4 L-AP4 DCG-IV LY379268 MAP4 L-SOP MSOP (RS)-PPG MPPG CPPG mGlu6L-AP4 DCG-IV L-SOP (RS)-PPG (S)-HomoAMPA mGlu7 L-SOP DCG-IV (RS)-PPGMPPG mGlu8 L-AP4 DCG-IV LY354740 MAP4 LY379268 L-SOP (RS)-PPG

MPEP can be prepared according to the chemical procedures described inWO99/02497 or purchased from Tocris, Ballwin, Mo. LY341495(2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionicacid) can be purchased from Tocris (Ballwin, Mo.). NBQX disodium(2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulphonamidedisodium) can be purchased from Tocris, Ballwin, Mo.

As used herein, an “effective amount” of an antagonist is an amount thatmodulates the normal activity of mGlu receptors 2, 3, or 5 in a subject.A “normal” activity of mGlu receptor represents a level of activity in acell or subject not having an mGluR-related disorder and can bedetermined using methods known in the art, some of which are disclosedherein. For example, LY341495 can be administered at a concentration ofabout 0.1-50 mg/kg, in certain aspects between 0.1 and 5 mg/kg. In someaspects of the invention, an effective amount of LY341 495 is at least0.5 mg/kg, for example, 0.5 mg/kg to about 10 mg/kg, or 0.5 mg/kg toabout 5 mg/kg. In certain aspects of the invention, LY341495 isadministered at a concentration of about 0.5 mg/kg or 1 mg/kg.

MPEP, in certain aspects of the invention, is administered in an amountof between about 0.01 and 25 mg/kg body weight. In certain aspects, MPEPis administered at a concentration equal to or greater than 1 mg/kg, forexample between about 3 and about 20 mg/kg. In other aspects, MPEP isadministered at a concentration of between about 5 and about 15 mg/kg.In other aspects, MPEP is administered at between about 7 and about 12mg/kg, for example at 9 mg/kg.

In one aspect of the invention, two or more antagonists are administeredat sub-effective amounts, below which one or more of the antagonists areeffective at treating the disorder on their own. For example, an mGluR5antagonist can be administered at a sub-effective amount and anmGluR2/mGluR3 antagonist can be administered at an effective amount, orvice versa. MPEP can be administered at 1 mg/kg or less and LY341495 canbe administered at an effective concentration, as indicated above. Forexample, MPEP can be administered at 0.1-3 mg/kg and LY341495 can beadministered at 0.5-5 mg/kg. In one aspect illustrated in Example 3,MPEP is administered at 1 mg/kg and LY341495 is administered at 0.5mg/kg. In addition, other specific aspects of the present inventioninclude the combination of 1 mg/kg LY341495 and 1 mg/kg MPEP; and 1mg/kg LY341495 and 9 mg/kg MPEP. As illustrated in Example 3, when usedin combination, an mGluR5 antagonist enhances the effectiveness intreating an addictive disorder, such as nicotine addiction, of anmGluR2/R3 antagonist. As also illustrated in Example 3, when used incombination, an mGluR2/3 antagonist enhances the effectiveness intreating an addictive disorder, such as nicotine addiction, of an mGluR5antagonist. It will be understood that the present invention provides abasis for further studies in humans to more precisely determineeffective amounts in humans. Doses used in the Examples section forrodent studies provide a basis for the ranges of doses indicated hereinfor humans and other mammals.

In certain aspects of the invention, for example, an amount of one ormore mGluR2, 3, and/or 5 antagonist is administered that is sufficientto diminish, inhibit, or eliminate desire for an addictive substancesuch as cocaine or nicotine. Furthermore, in certain aspects, an amountof an mGluR2, 3, and/or 5 antagonist is administered that is sufficientto diminish, inhibit, or eliminate the reinforcing properties of anaddictive substance, such as cocaine or nicotine. Furthermore, incertain aspects, an amount of an mGluR2 and/or 3 antagonist isadministered that is sufficient to diminish, inhibit, or eliminatedepression, such as the depression associated with withdrawal from anaddictive substance, such as cocaine or nicotine, or depressionassociated with drug use, or depression not associated with any druguse. Diminution, inhibition, and/or elimination of any of thesecharacteristics of the diseases targeted by the methods of the presentinvention indicate effective treatment of a metabotropic glutamatedisorder.

Disorders that can be effectively treated by modulating the activity ofmGluRs 2, 3, and 5, referred to herein as metabotropic glutamatedisorders, include addictive disorders and depression. Metabotropicglutamate disorders include disorders that involve one or both of aGroup I metabotropic glutamate receptor (mGluR) and one or both of aGroup II mGluR. Addictive metabotropic glutamate disorders include, forexample, nicotine addiction, alcohol addiction, opiate addiction,amphetamine addiction, cocaine addiction, methamphetamine addiction, andthe like.

It will be understood that the data provided in the Examples section forcertain drugs of abuse, such as cocaine and nicotine, is applicable toother drugs of abuse as well. It has been extensively hypothesized thatdependence on all major drugs of abuse is mediated by the sameneurobiological and behavioral mechanisms (Markou et al. 1998; Markou A& Kenny P J 2002 Neuroadaptations to chronic exposure to drugs of abuse:Relevance to depressive symptomatology seen across psychiatricdiagnostic categories, Neurotoxicity Research, 4(4), 297-313.; Koob & LeMoal, Neuropsychopharmacology, 24(2):97-129 2001; Barr, A. M., Markou,A. and Phillips, A. G. (2002) A “crash” course on psychostimulantwithdrawal as a model of depression, Trends in Pharmacological Sciences,23(1), 475-482, incorporated in its entirety by reference; Cryan, J. F.,Markou, A. and Lucki, I. (2002) Assessing antidepressant activity inrodents: Recent developments and future needs, Trends in PharmacologicalSciences, 23(5), 238-245). Thus, one would expect that a compoundeffective in treating one drug addiction is likely to be effective intreating another addiction also. For example, increase in serotonergicneurotransmission by co-administration of the selective serotoninreuptake inhibitor fluoxetine+the serotonin-1A receptor antagonistp-MPPI reversed the depression-like aspects of both amphetamine andnicotine withdrawal (Harrison, Liem & Markou, Neuropsychopharmacology2001; incorporated in its entirety by reference). Further, withdrawalfrom all major drugs of abuse (nicotine, cocaine, amphetamine, alcohol,opiates, phencyclidine) results in elevations in brain reward thresholdsreflecting a depression-like state (references in reviews Markou et al.1998; Markou & Kenny 2002; Barr et al. 2002; Cryan et al. 2002; andoriginal research reference for phencycline: Spielewoy, C. & Markou, A.2003, Withdrawal from chronic treatment with phencyclidine induceslong-lasting depression in brain reward function,Neuropsychopharmacology, 28, 1106-1116 and cocaine: Ahmed et al. 2002).Thus, treating such depression-like aspects of drug dependence andwithdrawal may assist people in abstaining from drug use.

In one aspect, the addictive disorder is nicotine addiction. In anotheraspect, the addictive disorder is cocaine addiction. In another aspect,the addictive disorder is alcohol addiction. In another aspect, theaddictive disorder is opiate addiction. In another aspect, the addictivedisorder is methamphetamine addiction. In another aspect, the addictivedisorder is amphetamine addiction.

In another aspect, the metabotropic glutamate disorder is depression.Example 1 illustrates that blockade of mGluR2/3 receptors hasantidepressant properties as reflected in reversal of the negativeaffective (depression-like) aspects of nicotine withdrawal. Thus,blockade of mGluR2 and mGluR3 reverses depression-like symptoms observedduring drug withdrawal, and possibly depression observed during drugdependence (Ahmed, S. H., et al. Nature Neuroscience, 5: 625-626 (2002),incorporated in its entirety by reference). Therefore, administration ofan effective amount of an antagonist of mGluR2 and mGlurR3 is likely tobe efficacious for treating non-drug-induced depressions, based on theknown neurobiological similarities mediating drug- and non-drug-induceddepressions (Markou et al. 1998, incorporated in its entirety byreference; Barr et al., 2002, incorporated in its entirety by reference;Cryan et al., 2002; incorporated in its entirety by reference; Harrisonet al., Neuropsychopharmacology, 25:55-71 (2001), incorporated in itsentirety by reference; Markou A and Kenny P J 2002, Neuroadaptations tochronic exposure to drugs of abuse: Relevance to depressivesymptomatology seen across psychiatric diagnostic categories,Neurotoxicity Research, 4(4), 297-313; incorporated in its entirety byreference). Additional observations further support the conclusion thatresults presented in Example 1 related to depression-like symptoms ofwithdrawal of an addictive substance, establish that antagonists ofmGluR2 and/or mGluR3 can be used to effectively treat non-drug-induceddepressions, as well. First, it has been shown that co-administration ofthe selective serotonin reuptake inhibitor fluoxetine and theserotonin-1A receptor antagonist p-MPPI, a clinically provenantidepressant drug treatment, reverses the depression-like aspects ofboth nicotine and amphetamine withdrawal (Harrison et al., (2001);incorporated in its entirety by reference). Second, as shown in Example4, co-administration of the selective serotonin reuptake inhibitorparoxetine and the serotonin-1A receptor antagonist p-MPPI, anotherclinically proven antidepressant drug treatment, also reversedamphetamine withdrawal. Third, bupropion, another clinically provenantidepressant treatment, reverses the depression-like aspects ofnicotine withdrawal (Cryan, J. F., et al., Psychopharmacology, 168,347-358 (2003); Example 5). Thus, clinically proven antidepressanttreatments reverse the depression-like aspects of drug withdrawal in themodel presented in Examples 1 and 4. Therefore, it can be inferred thata treatment (e.g., mGluR2/3 antagonist) that normalized thresholds inthe model, would be a clinically effective treatment. Further, thereversal of both amphetamine and nicotine withdrawal by the sameantidepressant treatment indicates that there are commonalities invarious types of depression, independent of what thedepression-induction mechanism is and/or the primary site of action ofthe drug of abuse (nicotinic receptor for nicotine, monoaminergictransporters for amphetamine).

The present invention in another embodiment, provides a method fortreating depressive symptoms and anxiety symptoms of depression. Themethod includes administering to a subject in need thereof, an effectiveamount of at least one antagonist which modulates mGluR2, mGluR3, andmGluR5, thereby treating the depressive symptoms and anxiety symptoms.In another aspect of this embodiment of the invention, at least oneantagonist of mGluR2 and/or R3 is administered during a depressed timeperiod, wherein the subject experiences symptoms of depression, whereasan mGluR5 antagonist is administered during time periods when thesubject experiences symptoms of anxiety. Depression is characterized byboth depressive symptoms and anxiety symptoms. Thus, the presentinvention, which provide an mGluR2/3/5 combination treatment provides aneffective antidepressant, with the mGluR2/3 antagonism amelioratingdepressive symptoms and the mGluR5 antagonist ameliorating anxietysymptoms. Accordingly, this embodiment takes advantage of theanti-depressive properties of mGluR2/3 antagonists and the anxiolyticproperties of mGluR5 antagonists (See e.g., Cosford, N. D., et al., J.Med. Chem. 16;46(2):204-6 (2003); Brodkin J., et al., Eur. J. Neurosci.,16(11):2241-4 (2002); and Brodkin J., et al., Pharmacol. Biochem. Behav.73(2):359-66 (2002)). Since anxiety is known to be a major symptom ofthe overall syndrome of depression, this embodiment of the invention iseffective at treating the anxiety symptoms of depression.

Depressive symptoms and anxiety symptoms are well known in the art.Methods of this embodiment of the invention treat one or more symptomsof depression and one or more symptoms of anxiety. Symptoms ofdepression, include, for example, but are not limited to, the following:a persistent sad, anxious or “empty” mood; sleeping too little orsleeping too much; reduced appetite and weight loss, or increasedappetite and weight gain; loss of interest or pleasure in activitiesonce enjoyed; restlessness or irritability; persistent physical symptomsthat don't respond to treatment (e.g., headaches, chronic pain, orconstipation and other digestive disorders); difficulty concentrating,remembering, or making decisions; fatigue or loss of energy; feelingguilty, hopeless or worthless; and thoughts of death or suicide.

Symptoms of anxiety include, but are not limited to, the following:

excessive worry, occurring more days than not, over a period of months,for example over a period of at least six months; unreasonable worryabout a number of events or activities, such as work or school and/orhealth; the inability to control worry; restlessness, feeling keyed-upor on edge; tiredness; problems concentrating; irritability; muscletension; and trouble falling asleep or staying asleep, or restless andunsatisfying sleep.

In certain aspects of embodiments of the present invention directed atmethods for treating a metabotropic glutamate disorder, the subject is amammalian subject, for example a human subject afflicted with ametabotropic glutamate receptor disorder, for example nicotineaddiction, cocaine addiction, or depression. The examples hereinillustrate the methods of the present invention in rodents. However, itwill be understood that the methods are expected to be efficacious inhuman subjects as well, due to the similarity between rodents and humansin the physiology of addictive disorders and depression (Markou et al.1998; Markou and Kenny 2002, incorporated in its entirety by reference),and the structure of mGlu receptors (Schoepp et al. Neuropharm. 1999,38, 1431-1476).; Schoepp D D (2001), J Pharmacol Exp Ther 299:12-20).

“Simultaneous administration” of two or more antagonists isadministration of the agonists to a subject within a short enough timeperiod such that a sufficient concentration of each of the antagonistsis present in the subject at the same time to modulate their respectivemGlu receptor targets. Therefore, it will be recognized that the maximumtime difference between administrations of antagonists that representsimultaneous administration depends on the half-life of the antagonistsadministered, the amount of antagonist administered, and the method andlocation by which the antagonists are administered, for example.

For certain aspects of the methods of the present invention, theantagonists are administered for periods of weeks, months, years, andpossibly indefinitely for subjects exhibiting failure to abstain fromdrug use or for chronic depressive disorders that may be unrelated todrug use or induced by drug use, but not remitting spontaneously withoutthe methods suggested herein.

The present invention, in another embodiment, provides a method forinhibiting drug-taking behavior, treating depression, and/or treatingthe depression-like state associated with drug use and dependence (Ahmedet al., 2002, incorporated herein in its entirety by reference), or withaddictive drug withdrawal, that includes administering to a subject inneed thereof, an effective amount of at least one antagonist whichmodulates mGluR2 and/or mGluR3, thereby treating consumption of theaddictive substance, depression, or the depression-like state of theaddictive drug dependence or drug withdrawal states. This embodiment isbased on the experimental evidence provided in Example 3 that treatmentwith an antagonist at mGlu2/3 receptors, such as LY341495, decreasesaddictive drug (e.g., nicotine) consumption in rats, as well as theevidence provided in Example 1 that treatment with an mGlu2/3 receptorantagonist, such as LY341495, can reverse the depression-like stateassociated with drug withdrawal. The addictive substance for example, isnicotine or cocaine. In certain aspects, the effective amount of atleast one antagonist is administered to decrease nicotine consumption.For example, in one aspect an effective amount of an antagonist ofmGluR2 and mGluR3, such as LY341495, is administered to decreasenicotine consumption. In certain aspects of the invention, an inhibitorof mGluR2 and/or mGluR3 is administered while a subject is experiencingwithdrawal. In another aspect of the invention, an inhibitor of mGluR2and/or mGluR3 is administered during a time period when a subject isactively using an addictive substance. In another aspect of theinvention, an inhibitor of mGluR2 and/or mGluR3 is administered during atime period when a subject is actively experiencing depressionassociated with drug use or not associated with drug use.

The present invention, in another embodiment, provides a method forantagonizing at least two of mGluR2, mGluR3, and mGluR5, that includessimultaneously administering to a subject in need thereof, an amount ofat least two antagonists that modulate at least two of mGluR2, mGluR3,and mGluR5. The amount of each antagonist is sufficient to modulate itstarget mGluR. This amount may be less than an effective amount of theantagonist when administered alone. However, the amount administered inthese embodiments is sufficient so that the combination of antagonistsis effective for treating a metabotropic disorder. In certain aspects,each antagonist is provided at an effective amount for treating ametabotropic glutamate disorder. In certain aspects, the subject isafflicted with depression, a nicotine addiction, or a cocaine addiction.In certain aspects, an antagonist that modulates mGluR2 and mGluR3 isadministered along with an antagonist that modulates mGluR5. Forexample, an effective amount of MPEP can be administered along with aneffective amount of LY341495. In another embodiment, a sub-effectiveamount of MPEP is administered along with an effective amount ofLY341495. In another embodiment, an effective amount of MPEP isadministered along with a sub-effective amount of LY341495. In anotherembodiment, a sub-effective amount of MPEP is administered along with asub-effective amount of LY341495.

The present invention in another embodiment provides a method fortreating an addictive disorder, also referred to herein as substanceabuse, that includes administering to a subject in need thereof, aneffective amount of at least one antagonist that modulates at least oneof mGluR2, 3, and 5 during a first time period, followed byadministering at least one antagonist that modulates at least one ofmGluR2 and/or 3 during a second time period. The first time period, forexample, is a time period wherein the subject expects to be in anenvironment wherein, or exposed to stimuli in the presence of which, thesubject habitually uses an addictive substance, or wherein the subjectis actively using the addictive substance. The second time period, forexample, is a time period wherein the subject is suffering fromwithdrawal and/or depression. One aspect of this embodiment of theinvention, for example, includes administering MPEP and/or LY341495during a first time period, and administering LY341495 during awithdrawal or depression period.

This embodiment is based on the finding presented in the Examples hereinthat administration of an antagonist of mGluR5 and/or an antagonists ofmGluR213 decrease self-administration of an addictive drug such asnicotine or cocaine. Furthermore, this embodiment is based on thefinding that administration of an antagonist of mGluR2/3 reverses atleast some of the negative effects of nicotine withdrawal. In one aspectof this embodiment, an antagonist of mGluR5 and an antagonist of mGluR2and/or mGluR3 are administered and the depression-like symptoms of drugdependence and withdrawal are monitored. If withdrawal symptoms ordepression symptoms become too intense, administration of the mGluR5antagonist can be terminated at least temporarily, while the mGluR2/3antagonist can continue to be administered.

In certain embodiments of the invention, an addictive or depressivedisorder is treated by administering an effective amount of anAMPA/Kainate receptor agonist or partial agonist to a subject. Incertain aspects of this embodiment, one or more agonists or partialagonists are administered to the subject that modulate the AMPA/Kainatereceptor as well as at least one of the mGluR2, mGluR3, and mGluR5receptors. Typically, one agonist or partial agonist of the AMPA/Kainatereceptor is administered along with at least one antagonist of mGluR2,mGluR3, and/or mGluR5. In certain embodiments one or more agonists orpartial agonists are administered that modulate the AMPA/Kainatereceptor and antagonists that modulate the mGluR5, for example byadministration of AMPA/Kainate agonists or partial agonists and MPEP. Inanother embodiment one or more agonists or partial agonists areadministered that modulate the AMPA/Kainate receptor, along with one ormore antagonists that modulate mGluR2 and/or mGluR3.

AMPA/Kainate receptor agonists or partial agonists include, for example,CPCCOet (7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethylester (Litschig S., et al., Molecular Pharmacology 55(3):453-561(1999)). The use of AMPA/Kainate receptor partial agonists provides theadvantage over AMPA/Kainate agonists, that the partial agonistsgenerally have better side-effect profiles.

The route of delivery of the antagonists or agonists employed byinvention methods is determined by the particular disorder. Antagonistsor agonists may be delivered orally, intravenously, intraperitoneally,intramuscularly, subcutaneously, intranasally, and intradermally, aswell as, by transdermal delivery (e.g., with a lipid-soluble carrier ina skin patch placed on skin), or even by gastrointestinal delivery(e.g., with a capsule or tablet). Furthermore, antagonists or agonistsused in the methods of the present invention, in certain aspects aredelivered directly to the brain or certain regions of the brain toactivate or inhibit receptors at specific brain sites producing thedesirable effect without inhibiting or activating receptors at otherbrain sites, thus avoiding undesirable side-effects or actions that maycounteract the beneficial therapeutic action mediated by the formersite(s). The dosage will be sufficient to provide an effective amount ofan antagonist either singly or in combination, as discussed above. Somevariation in dosage will necessarily occur depending upon the conditionof the patient being treated, and the physician will, in any event,determine the appropriate dose for the individual patient. The dose willdepend, among other things, on the body weight, physiology, and chosenadministration regimen.

The antagonists employed in invention methods are administered alone orin combination with pharmaceutically acceptable carriers, in eithersingle or multiple doses. Suitable pharmaceutical carriers include inertsolid diluents or fillers, sterile aqueous solutions, and variousnontoxic organic solvents. The pharmaceutical compositions formed bycombining one or more antagonist with the pharmaceutically acceptablecarrier are then readily administered in a variety of dosage forms suchas tablets, lozenges, syrups, injectable solutions, and the like. Thesepharmaceutical carriers can, if desired, contain additional ingredientssuch as flavorings, binders, excipients, and the like. Thus, forpurposes of oral administration, tablets containing various excipientssuch as sodium citrate, calcium carbonate, and calcium phosphate areemployed along with various disintegrants such as starch, and preferablypotato or tapioca starch, alginic acid, and certain complex silicates,together with binding agents such as polyvinylpyrolidone, sucrose,gelatin, and acacia. Additionally, lubricating agents, such as magnesiumstearate, sodium lauryl sulfate, and talc are often useful for tabletingpurposes. Solid compositions of a similar type may also be employed asfillers in salt and hard-filled gelatin capsules. Preferred materialsfor this purpose include lactose or milk sugar and high molecular weightpolyethylene glycols.

When aqueous suspensions of elixirs are desired for oral administration,the antagonists may be combined with various sweetening or flavoringagents, colored matter or dyes, and if desired, emulsifying orsuspending agents, together with diluents such as water, ethanol,propylene glycol, glycerin, and combinations thereof. For parenteraladministration, solutions of preparation in sesame or peanut oil or inaqueous polypropylene glycol are employed, as well as sterile aqueoussaline solutions of the corresponding water soluble pharmaceuticallyacceptable metal salts previously described. Such an aqueous solutionshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic with sufficient saline or glucose. These particularaqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal injection. The sterileaqueous media employed are all readily obtainable by standard techniqueswell known to those skilled in the art.

In another embodiment, the present invention provides a method ofscreening for an agent that improves the ability of a known inhibitor toat least partially normalize intracranial self-stimulation (ICSS)threshold and/or improves the ability of a known inhibitor to inhibitconsumption of an addictive substance for a non-human, mammaliansubject. The method includes:

-   -   a) affecting the ICSS threshold of the subject;    -   b) administering to the subject, a sufficient amount of the        known inhibitor to inhibit consumption of an addictive substance        and/or at least partially normalize the ICSS threshold when        administered alone or in combination with another inhibitor,        wherein the known inhibitor is an antagonist of mGluR2 and/or        mGluR3 and/or mGluR5;    -   c) administering to the subject, an effective amount of a test        agent, wherein the test agent is a known or suspected antagonist        of mGluR2 and/or mGluR3 and/or mGluR5; and    -   d) determining whether the test agent improves the ability of        the known inhibitor, to at least partially normalize the ICSS        threshold, and optionally to inhibit one or both of consumption        of the addictive substance, thereby identifying an agent that        improves the ability of the known inhibitor to normalize ICSS        threshold and/or improves the ability of a known inhibitor to        inhibit consumption of an addictive substance, or,        alternatively, determining whether the test agent improves the        ability of the known inhibitor, to at least decrease consumption        of an addictive substance, and optionally to partially normalize        the ICSS threshold or both thereby identifying an agent that        improves at least the ability of the known inhibitor to inhibit        consumption of an addictive substance and/or normalize ICSS        threshold.

Intracranial self-stimulation (ICSS) thresholds are at least partiallynormalized when an increase or decrease on ICSS threshold caused by ametabotropic glutamate disorder is at least partially inhibited (i.e.adjusted back to the ICSS threshold of a subject that is not afflictedwith the metabotropic glutamate disorder). For example, withdrawal fromchronic nicotine administration (Kenny et al., 2003) or chronicself-administration of cocaine (Ahmed et al. 2002, incorporated in itsentirety by reference) are known to elevate an ICSS threshold (see e.g.,Kenny et al. 2003). Therefore, partial normalization of an ICSSthreshold during cocaine administration or during nicotine withdrawalwill lower the ICSS threshold from the elevated threshold, reflecting adepressed state, normally found during chronic cocaine administration ornicotine withdrawal, closer to that found in a normal subject.Accordingly, methods of this embodiment of the invention can utilizechronic administration of an addictive substance (e.g., cocaine) ortermination of administration of an addictive substance (e.g., nicotine)to affect (i.e., denormalize) the ICSS threshold before or duringadministration of the known inhibitor and the test agent. Acuteadministration of an addictive substance typically decreases the ICSSthreshold, while chronic administration of an addictive substance and/ortermination of administration of an addictive substance typicallyincreases the ICSS threshold.

In addition to the methods above, the ICSS threshold can be affected byother known methods. For example, the chronic mild stress procedure canbe used to induce threshold elevations reversible by antidepressanttreatments (Moreau J. L., Bos M., Jenck F., Martin J. R., Mortas P. andWichmann J. (1996), Eur Neuropsychopharmacology, 6, 169-175; Moreau J.L., Bourson A., Jenck F., Martin J. R. and Mortas P. (1994), JPsychiatry Neurosci 19, 51-56; Moreau J. L., Jenck F., Martin J. R.,Mortas P. and Haefely W. E. (1992), Eur Neuropsychophartnacoogy,2,43-49).

Methods for self-administration of an addictive agent and for analyzingICSS are disclosed in the Examples section. The methods of the presentinvention not only can be used to identify an agent that is effective atimproving the effectiveness of a known inhibitor, but also can be usedto determine which of the characteristics/symptoms/aspects associatedwith substance abuse or withdrawal are effected by the test agent, asillustrated in the Examples, and discussed further in the followingparagraphs.

Methods analyzing consumption of an addictive substance can be performedunder a self-administration fixed ratio schedule of reinforcement orunder a progressive schedule of reinforcement, examples of which areprovided in Examples 2 and 3. In a fixed ration schedule, an animalresponds a ‘fixed’ number of times on an active lever to obtain a druginfusion. Fixed ratio (FR) schedules of reinforcement provide importantinformation as to whether a drug is reinforcing. In contrast, under aprogressive ratio (PR) schedule of reinforcement, each time an animalresponds on the active lever to receive a drug infusion, the number oftimes that the animal must subsequently respond to receive the nextinfusion is progressively increased. By determining how hard an animalis willing to work for a drug injection, while limiting total intake,the PR schedule allows better separation of motivation for drugconsumption from possible satiating effects of cumulative drug doses(Stafford et al. 1998).

These characteristics result in theoretically different interpretationsof the factors controlling drug-seeking and drug-taking behavior on a PRcompared with a FR schedule of reinforcement. For example, someresearchers have suggested that FR schedules measure the pleasurable orhedonic effects of a drug (McGregor and Roberts 1995; Mendrek et al.1998), whereas PR schedules provide a better measure of the incentive orthe ‘motivation’ to obtain a drug (Markou et al., 1993). Afteracquisition of cocaine or nicotine self-administration under an FRschedule, as described above, rodents, such as rats or mice, can beswitched to a PR schedule of reinforcement in which an increasingsequence of level presses can be required to receive each subsequentinfusion of an addictive substance such as nicotine or cocaine, ortrained from the beginning on the PR schedule For example, 5, 10, 17,24, 32, 42, 56, 73, 95, 124, 161, 208, etc. level presses can berequired to receive each subsequent infusion under a progressive ratioschedule of reinforcement. In certain aspects, a test agent can beadministered to a non-human subject for a period of time, for example,15 minutes, 30, minutes, 45 minutes, 1 hour, etc. before the subject isplaced in a PR of FR schedule of reinforcement session. The session canbe conducted for a fixed period of time (e.g., 1 hour, 2 hours, 3 hours,4 hours, etc.). A break-point can be determined for each session.Break-point is defined as the highest ratio (i.e., the highest number oflever presses emitted for a drug injection within the time periodallowed, usually 1 hour since the last drug injection was earned)achieved before the session is terminated; the session is terminated ifthe subject failed to earn a drug infusion during one hour.

Screening methods of the invention can also be used to determine whethera test agent reduces the hedonic actions of cocaine. Cocaine-inducedlowering of ICSS thresholds represents an accurate measure of cocaine'shedonic and euphorigenic actions. Thus, to test the hypothesis that atest agent attenuates the hedonic actions of cocaine, a test agent'seffect on cocaine-induced lowering of ICSS thresholds can be determined.Cocaine can be used at an amount sufficient to lower an ICSS threshold,for example 10 mg/kg, without affecting performance in the ICSSprocedure (Kenny et al., 2002b; Markou & Koob 1992).

Furthermore, depression-like symptoms of withdrawal and drug dependencecan be measured by measuring the increase in ICSS reward threshold thatoccurs after termination of administration of an addictive substance andduring (or closely timely related, such as immediately before and/orafter) drug consumption. Therefore, a test agent's ability to inhibitthese depression-like symptoms can be measured by determining whetherthe test agent at least partially inhibits the increase in ICSS rewardthreshold that occurs during drug use or withdrawal (i.e. normalized thethreshold). Methods for determining ICSS thresholds are known in the artand disclosed in the Example section. Therefore, screening methods ofthis embodiment of the invention not only identify agents that improvethe inhibition by a known inhibitor, they also can be used to identifythe specific characteristics of an addiction (e.g., hedonic effects of adrug, motivation to take a drug, depression associated with drugdependence and/or following drug withdrawal) that are improved by thetest agent.

In certain aspects of this embodiment, a test agent known or suspectedto antagonize mGluR2 and/or mGluR3 is analyzed in combination with aknown inhibitor that is an antagonist of mGluR5. Conversely, in otheraspects of this embodiment, a test agent known or suspected toantagonize mGluR5, is analyzed with a known inhibitor that is anantagonist of mGluR2 and/or mGluR3. In certain aspects, the knowninhibitor is MPEP and the test agent is known or suspected of being anantagonist of mGluR2 and/or mGluR3. In other aspects, the knowninhibitor is LY341495 and the test agent is known or suspected of beingan antagonist of mGluR5.

For screening embodiments of the invention, the subject is a non-humanmammalian subject, for example a primate or a rodent, such as a mouse ora rat. The non-human subject is afflicted with a metabotropic glutamatereceptor disorder, for example nicotine addiction, cocaine addiction, ordepression. The disease can be induced using methods known in the art,as illustrated by the Examples, or by using lines of organisms that areknown to be at increased risk for developing the disorder.

The term “test agent” is used herein to mean any agent that is beingexamined for the ability to improve the ability of the known inhibitorto inhibit consumption of an addictive substance or normalize ICSSthresholds. The method generally is used as a screening assay toidentify molecules that can act as a therapeutic agent for treatingdepressive disorders or addictive disorders such as cocaine addiction ornicotine addiction. As indicated above, the test agent can be an agentknown to inhibit mGluR2, mGluR3, and/or mGluR5. Furthermore, thescreening methods of the present invention can be combined with othermethods that analyze test agents for the ability to antagonize mGluR2,mGluR3, and/or mGluR5. For example, a cell based high throughput assaycan be used to screen for test agents that are antagonists for mGluR2,mGluR3, and/or mGluR5, using methods known in the art. Test agentsidentified as antagonists of mGluR2, mGluR3, and/or mGluR5 can then beanalyzed in the screening method provided in this embodiment of theinvention, to determine whether the test agents improve the ability ofthe known inhibitor to inhibit consumption of an addictive substance oraffect ICSS reward thresholds.

A test agent can be any type of molecule, including, for example, apeptide, a peptidomimetic, a polynucleotide, or a small organicmolecule, that one wishes to examine for the ability to act as atherapeutic agent, which is an agent that provides a therapeuticadvantage to a subject receiving it.

In another embodiment, the present invention provides kits that areuseful for carrying out the methods of the present invention. Thecomponents of the kits depend on the specific method that is intended tobe performed by the kit. For example, the kit can be useful for carryingout a method to treat a metabotropic glutamate disorder. In this aspect,the kit can include at least one container that contains an antagonistthat modulates metabotropic glutamate receptor 2 (mGluR2), mGluR3,and/or mGluR5. In one aspect, the kit includes a first container with aninhibitor of mGluR5 and a second container with an inhibitor of mGluR2and/or mGluR3. In one aspect, the kit includes a container of MPEP and acontainer of LY341495. The antagonists included in the test kit areprovided in an amount and form that is sufficient to allow an effectiveamount to be administered to the subject. The kit for example, can alsoinclude instructions regarding effective use of the antagonists in thetreatment of substance abuse and depression. The kit in certain aspectsincludes information that is generally useful for a depressed individualor an individual suffering from an addiction.

In another aspect, the kits can be useful for screening for an agentthat improves the ability of a known inhibitor to inhibit consumption ofan addictive substance or at least partially normalize intracranialself-stimulation threshold. In this aspect, the kit for example,includes a container having a known inhibitor. Furthermore, the kit mayinclude a container of an addictive substance to be administered to anon-human mammalian subject.

The present invention in another embodiment provides a method fortreating an addictive disorder, that includes administering to a subjectin need thereof, an effective amount or sub-effective amount ofbupropion during a first time period, and administering of bupropionduring a second time period. The first time period, for example, is atime period wherein the subject expects to be in an environment wherein,or exposed to stimuli in the presence of which, the subject habituallyuses an addictive substance, or wherein the subject is actively usingthe addictive substance. The second time period, for example, is a timeperiod wherein the subject is suffering from withdrawal and/ordepression. In one aspect of this embodiment, in the first time periodbupropion is administered at a lower dose than during the second period.

This embodiment is based on the finding presented in Example 5 thatlower doses, in fact sub-effective doses (5 mg/kg) of bupropion wereeffective at blocking the threshold lowering effects of acute nicotineadministration, and higher concentration (10-40 mg/kg) of bupropionadministration normalizes ICSS thresholds that were affected by nicotineadministration and/or withdrawal. A sub-effective dose of bupropion is adose that on its own does not lower brain reward thresholds, but that iscapable of completely reversing the reward-enhancing effects of acutenicotine.

In another aspect of this embodiment of the invention, the presentinvention provides a method for treating addictive drug dependenceand/or withdrawal and/or depression associated with drug use or drugwithdrawal that includes administering to a subject in need thereof, aneffective amount of a selective serotonin reuptake inhibitor, such asparoxetine, and a serotonin-1A receptor antagonist. The serotonin-1Areceptor antagonist, in certain aspects of the invention, is p-MPPI.Another serotonin-1A receptor antagonist that can be used is pindolol.This embodiment is based on the results presented in Example 4.

In another embodiment the present invention provides a method fortreating an addictive disorder that includes administering to a subjectin need thereof, an effective amount of a selective serotonin reuptakeinhibitor, such as paroxetine, a serotonin-1A receptor antagonist,and/or bupropion, and administrating to the subject an effective amountof an antagonist of mGluR2, mGluR3, and/or mGluR5. In certain aspects ofthis embodiment, where an mGluR5 antagonist is administered, the mGluR5administration is stopped during periods wherein the subject suffersfrom withdrawal. In certain aspects, administration of the selectiveserotonin reuptake inhibitor, a 5-HT1A receptor antagonist, and/orbupropion is commenced during periods wherein the subject suffers fromwithdrawal.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLE 1 Group II Metabotropic and AMPA/Kainate Glutamate ReceptorsRegulate the Deficit in Brain Reward Function Associated with NicotineWithdrawal in Rats

This example illustrates that an antagonist of mGluR2 and mGluR3receptors and possibly an agonist of AMPA/Kainate glutamate receptorscan lower the deficit in brain reward function associated with nicotinewithdrawal in rats. Nicotine withdrawal precipitates an aversiveabstinence syndrome in human smokers hypothesized to provide animportant source of motivation contributing to the persistence of thesmoking habit and relapse during abstinence (Kenny and Markou, 2001).The data provided in this example strongly suggest a role for Group IImetabotropic glutamate receptors in generating the reward deficitsassociated with nicotine withdrawal by demonstrating that activation ofmGluII receptors precipitated ICSS threshold elevations innicotine-dependent rats similar to those observed during spontaneousnicotine withdrawal. Further, activation of mGluII receptors in the VTAalso elevated thresholds in nicotine-dependent rats, providing furthersupport for an important role of the VTA in mediating the actions ofnicotine on reward pathways. Consistent with the above, blockade ofmGluII receptors attenuated the reward deficits in rats undergoingspontaneous nicotine withdrawal. Finally, the data provided in thisexample also strongly suggest a role for AMPA/kainate metabotropicglutamate receptors in generating the reward deficits associated withnicotine withdrawal by demonstrating that antagonism of AMPA/kainitereceptors precipitated ICSS threshold elevations in nicotine-dependentrats similar to those observed during spontaneous nicotine withdrawal.

Materials and Methods

Subjects

Subjects were 149 male Wistar rats weighing 300-320 g at the start ofeach experiment. Rats were obtained from Charles River Laboratories(Raleigh, N.C.) and were housed with food and water available adlibitum. Animals were maintained in a temperature-controlled vivariumunder a 12 hr light/dark cycle (lights off at 10:00 am). In each caseanimals were tested during the dark portion of the light/dark cycle,except for the spontaneous nicotine withdrawal experiment when rats weretested at time-points according to the experimental design.

Drugs

(−)-Nicotine hydrogen tartrate salt ((−)-1-methyl-2-(3-pyridyl)pyrrolidine) and (+)-MK-801 hydrogen maleate (5R,10S)-(+)-5-Methyl-10,11 -dihydro-5H-dibenzo(a,d)cyclohepten-5,10-iminehydrogen maleate) were purchased from Sigma Chemical Co., St. Louis,Mo.; LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid) and NBQX disodium(2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulphonamidedisodium) were purchased from Tocris, Ballwin, Mo. LY314582 (the racemicmixture of LY354740 ((+)-2-aminobicyclo(3.1.0)hexane-2,6-dicarboxylicacid)) and MPEP (2-methyl-6-(phenylethynyl)-pyridine) were synthesized.CGP44532 (3-Amino-2-(S)-hydroxypropyl-methyl-phosphinic acid) wasgenerously provided by Novartis Pharma AG. Drugs were preparedimmediately before each administration. For systemic administration, alldrugs were dissolved in sterile water and administered byintraperitoneal injection, in a volume of 1 mil/kg body weight, 30 minbefore the experimental session. For direct intra-VTA administration,LY314582 was dissolved in artificial cerebrospinal fluid (aCSF) of thefollowing composition (in mM): 126.6 NaCl, 27.4 NaHCO₃, 2.4 KCI, 0.5KH₂PO₄, 0.89 CaCl₂, 0.8 MgCl₂, 0.48 Na₂HPO₄ and 7.1 glucose, pH 7.4.Rats received intra-VTA injections immediately prior to the initiationof the experimental session. Unless otherwise stated, drug doses referto the salt form.

Apparatus

Intracranial self-stimulation training and testing took place in sixteenPlexiglas operant chambers (25×31×24 cm) (Med Associates, St. Albans,Vt.). One wall contained a metal wheel manipulandum that required 0.2 Nforce to rotate it one-quarter of a turn. The wheel (5 cm in width)extended out of the wall -3 cm. Intracranial stimulation was deliveredby constant current stimulators. Subjects were connected to thestimulation circuit through flexible bipolar leads attached togold-contact swivel commutators mounted above the chamber. Thestimulation parameters, data collection, and all test session functionswere controlled by a microcomputer.

Surgery

Placement of electrodes and cannulas. Rats were anaesthetized byinhalation of 1-3% halothane in oxygen and positioned in a stereotaxicframe (Kopf Instruments, Tujunga, Calif.). The incisor bar was adjustedto 5 mm above the interaural line, and the skull exposed. Stainlesssteel bipolar electrodes (11 mm in length) were implanted into theposterior lateral hypothalamus (AP: −0.5 mm from bregma; ML: ±1.7 mm;DV: 8.3 mm from dura). For the VTA infusion experiment, bilateralstainless steel guide cannulas were implanted 3 mm above the VTA (AP:−3.2 mm from bregma; ML: ±1.7 mm; DV: 5.3 mm from skull surface; angleof 10° from midline), at the same time that ICSS electrodes wereimplanted. Cannulas were kept patent using 14 mm long stainless steelstylets (30 gauge). Animals were allowed to recover from surgery for atleast 7 days prior to training in the ICSS paradigm.

Osmotic Mini-Pump Surgery.

Rats were anaesthetized by inhalation of 1-3% halothane in oxygen andprepared with Alzet osmotic mini-pumps (model 2ML4 (28 day); AlzaCorporation, Palo Alto, Calif.) placed subcutaneously (back of theanimal parallel to the spine). Pumps were filled with either sterilewater or nicotine salt solution. The concentration of the nicotine saltsolution was adjusted according to animal body weight, resulting indelivery of 9 mg/kg/day (3.16 mg/kg, free base). This dose of nicotinemaintains stable plasma levels (˜44 ng/ml) comparable to those obtainedin human smokers consuming approximately 30 cigarettes per day(Benowitz, 1988, N Engl J Med 319:1318-1330). Following mini-pumpimplantation (or removal), the surgical wound was closed with 9 mmstainless steel wound clips and treated with topical antibiotic(Bacitracin) ointment.

Intracranial Self-Stimulation Reward Threshold Procedure

Animals were trained to respond according to a modification of thediscrete-trial current-threshold procedure of Kornetsky and Esposito(Fed Proc 38:2473-2476, 1979), which has been described in detailpreviously (Markou and Koob, 1992 Physiol Behav 51:111-119). Briefly, atrial was initiated by the delivery of a non-contingent electricalstimulus. This electrical reinforcer had a train duration of 500 ms andconsisted of 0.1 ms rectangular cathodal pulses that were delivered at afrequency of 50-100 Hz. The current intensity delivered was adjusted foreach animal and typically ranged from 50 to 200 μA. A one-quarter wheelturn within 7.5 sec of the delivery of the non-contingent electricalstimulation resulted in the delivery of an electrical stimulus identicalin all parameters to the non-contingent stimulus that initiated thetrial. After a variable inter-trial interval (7.5-12.5 sec, average of10 sec), another trial was initiated with the delivery of anon-contingent electrical stimulus. Failure to respond to thenon-contingent stimulus within 7.5 sec resulted in the onset of theinter-trial interval. Responding during the inter-trial interval delayedthe onset of the next trial by 12.5 sec. Current levels were varied inalternating descending and ascending series. A set of three trials waspresented for each current intensity. Current intensities were alteredin 5 μA steps. In each testing session, four alternatingdescending-ascending series were presented. The threshold for eachseries was defined as the midpoint between two consecutive currentintensities that yielded “positive scores” (animals responded for atleast two of the three trials) and two consecutive current intensitiesthat yielded “negative scores” (animals did not respond for two or moreof the three trials). The overall threshold of the session was definedas the mean of the thresholds for the four individual series. Eachtesting session was ˜30 min in duration. The latency between the onsetof the non-contingent stimulus and a positive response was recorded asthe response latency. The response latency for each test session wasdefined as the mean response latency of all trials during which apositive response occurred. After establishment of stable ICSS rewardthresholds, rats were tested in the ICSS procedure once daily except forthe spontaneous nicotine withdrawal experiment when rats were tested attime-points according to the experimental design.

Intracerebral Injection Procedure

All injections were administered bilaterally in a volume of 0.5 μl/sidegiven over 66 sec through 17 mm injectors. The injectors were connectedto calibrated polyethylene-10 tubing preloaded with drug solution andprotruded 3 mm below the ends of the cannulas into the VTA. Afterinfusion, the injectors were kept in place for an additional 60 sec toallow for drug diffusion. Injectors were then removed and replaced with14 mm wire stylets, and the animals then placed directly into the ICSStesting apparatus. Injections were made using a Harvard microinfusionpump (Model 975).

Experimental Design

Systemic administration experiments. These experiments investigatedwhether nicotine withdrawal, as measured by elevations in ICSSthresholds, could be precipitated in nicotine-treated rats by systemicadministration of an agonist at mGluII receptors (LY314582), an agonistat GABA_(B) receptors (CGP44532), or antagonists at mGlu5 (MPEP), NMDA(MK-801) or AMPA/Kainate (NBQX) glutamate receptors. For each drugtested, rats were trained in the ICSS paradigm until stable baselineresponding was achieved, defined as ≦10% variation in thresholds for 3consecutive days and requiring approximately 14 days of daily testing.In each case, drug-naïve rats were then allocated to two separate groupssuch that there was no difference in mean baseline ICSS thresholds orbody weight between groups. One group was then prepared withsubcutaneous osmotic mini-pumps delivering vehicle and the second groupwith mini-pumps delivering 9 mg/kg/day nicotine hydrogen tartrate (3.16mg/kg/day nicotine free base). There was a minimum seven-day intervalafter mini-pump implantation, during which ICSS reward thresholdscontinued to be measured daily, before the effect of any systemicallyadministered drug on reward thresholds was evaluated. This time periodwas sufficient to produce robust elevations in thresholds innicotine-treated but not vehicle-treated rats upon abrupt removal ofmini-pumps (i.e. spontaneous withdrawal) or administration of nicotinicreceptor antagonists (i.e. precipitated withdrawal) (Malin et al., 1992,Pharmacol Biochem Behav 43:779-784; Malin et. al., 1994,Psychopharmacology 115:180-184; Hildebrand et al., 1997,Psychopharmacology 129:348-356; Hildebrand et al., 1999,Neuropsychopharmacology 21:560-574; Epping-Jordan et al., 1998, Nature393:76-79; Watkins et al., 2000, J Pharmacol Exp Ther 292:1053-1064).Separate groups of nicotine-treated rats and their correspondingnicotine-naïve control group were then injected intraperitoneally withthe mGluII receptor agonist LY314582 (0, 2.5, 0.5, 7.5 mg/kg; n=9nicotine, n=11 control), the GABA_(B) receptor agonist CGP44532 (0,0.065, 0.125, 0.25, 0.5 mg/kg; n=5 nicotine, n=5 control), the mGlu5receptor antagonist MPEP (0, 0.01, 0.05, 0.1 mg/kg; n=8 nicotine, n=7vehicle or 0, 0.5, 1, 2, 3 mg/kg; n=13 nicotine, n=13 vehicle), the NMDAreceptor antagonist MK-801 (0, 0.01, 0.05, 0.1, 0.175, 0.2 mg/kg; n=10nicotine, n=9 control) or the AMPA/Kainate receptor antagonist NBQX (0,0.01, 0.025, 0.05, 0.075, 0.1, 0.5, 1 mg/kg; n=10 nicotine, n=12control) according to a within-subjects Latin square design and ICSSthresholds evaluated 30 min later. A minimum of 48 h were allowedbetween each injection in the Latin square design, during which ICSSthresholds continued to be measured, to ensure that ICSS thresholdsreturned to baseline. The doses of LY314582 and MPEP were chosen basedon a previous study demonstrating that ≧10 mg/kg LY314582 and ≧3 mg/kgMPEP elevated ICSS thresholds in drug-naïve rats (Harrison et al., 2002,Psychopharmacology 160:56-66). The doses of CGP44532 were chosen basedon a previous study demonstrating that ≧0.25 mg/kg elevated ICSSthresholds in drug-naïve rats (Macey et al., 2001, Neuropharmacology40:676-685). For the potential demonstration of interaction effects itwas important to include doses of the test drugs that did not alterthresholds under baseline conditions.

Intra-Ventral Tegmental Area LY314582 Experiment.

After stable baseline ICSS responding was achieved (≦10% variation inthreshold for 3 consecutive days), rats (n=15) with bilateral cannulasdirected toward the VTA were allocated to two groups such that therewere no differences in mean baseline reward thresholds or body weightbetween groups. One group was then prepared with subcutaneous osmoticmini-pumps delivering vehicle and the second group with mini-pumpsdelivering nicotine (3.16 mg/kg/day nicotine free base). Animals againwere tested in the ICSS paradigm each day for seven days prior to drugtreatment. Both groups of rats were then injected directly into the VTA,as described above, with LY314582 (0, 10, 50 and 100 ng/side; n=7nicotine, n=8 control) according to a within-subjects Latin squaredesign, and ICSS reward thresholds were evaluated immediatelypost-injection. There was a minimum 48 h interval between eachinjection, during which ICSS thresholds continued to be measured, toallow thresholds to return to baseline levels before further drug tests.At the conclusion of the experiment, all animals were anaesthetized andtheir brains removed and immediately placed on ice. The brains were cutin 50 μm sections, and placements of the injectors and the electrodeswere examined (see FIG. 1 for histological verification of injectionsites). Only those rats with injection tips located within the VTA wereincluded in statistical analyses.

Spontaneous Nicotine Withdrawal Experiment.

Osmotic mini-pumps were surgically removed from nicotine-treated rats(n=15) (defined as rats having been prepared with mini-pumps delivering3.16 mg/kg/day nicotine free-base for at least seven days) orcorresponding control rats (n=17; rats prepared with vehicle-containingmini-pumps). All rats were then tested in the ICSS procedure at 12, 18,24, 36, 48 and 72 hr after the removal of osmotic mini-pumps. These timepoints were chosen based on the time course of threshold elevationspreviously observed during spontaneous nicotine withdrawal after removalof nicotine-delivering osmotic mini-pumps (Harrison et al., 2001,Neuropsychopharmacology 25:55-71; K.L.). Based on the ICSS rewardthresholds obtained at the 12 h time-point, nicotine-withdrawing ratswere allocated to two groups such that there was no difference in themagnitude of reward threshold elevations between each group(117.67±3.1%, n=8; 119.93±3.5%, n=7). Similarly, control rats wereallocated to two groups such that there was no difference in mean rewardthresholds between these groups (106.45±5.2%, n=7; 103.63±3.6%, n=10).Thirty min before being tested at the 18 h time-point, one group ofnicotine withdrawing and one group of control rats were injected withLY341495 (1 mg/kg); the remaining rats were injected with vehicle.

Statistical Analyses

For all experiments, except the spontaneous nicotine withdrawalexperiment, percentage change from baseline reward threshold wascalculated by expressing the drug-influenced threshold scores as apercentage of the previous days threshold (i.e. a drug-free baselinethreshold). These percentages of baseline scores were subjected totwo-factor repeated measures analyses of variance (ANOVA), withtreatment drug dose as the within-subjects factor and pump content(nicotine or control) as the between-subjects factor. For thespontaneous nicotine withdrawal experiment, percentage change frombaseline reward threshold was calculated by expressing the thresholdscores obtained at each time-point during withdrawal as a percentage ofthresholds for each rat on the day immediately prior to mini-pumpremoval. These percentages of baseline scores were subjected tothree-factor repeated measures ANOVA. The within-subjects factor was thetime after mini-pump removal, and the two between-subjects factors werepump content (nicotine or vehicle) and acute drug treatment (LY314582 orvehicle). For all experiments, response latency data were analyzed inthe same manner as the threshold data. After statistically significanteffects in the ANOVAs, post-hoc comparisons among means were conductedwith the Fisher's LSD test.

Results

Intraperitoneal administration of the mGluII receptor agonist LY314582(2.5-7.5 mg/kg) elevated ICSS reward thresholds in nicotine-treated butnot control rats. This effect was reflected in a statisticallysignificant effect of group (F_((1,18))=7.43, p<0.05), a significanteffect of dose (F_((3,54))=5.02, p<0.005), and a significant group×doseinteraction (F_((3,54))=2.79, p<0.05). Post-hoc analysis revealed thatthe highest dose of LY314582 (7.5 mg/kg) elevated reward thresholds innicotine-treated rats compared to vehicle treatment (p<0.01), andcompared to control rats tested with the same dose (p<0.01) (FIG. 2A).In contrast to its effects on reward thresholds, LY314582 had no effecton response latencies in nicotine-treated or control rats(F_((3,54))=0.59, NS) at any dose tested (FIG. 2B).

As shown in Table 1, the selective GABA_(B) receptor agonist CGP44532did not precipitate reward threshold elevations in nicotine treated ratsat the lowest doses tested (0.065-0.25 mg/kg), whereas at the highestdose tested, CGP44532 (0.5 mg/kg) elevated reward thresholds innicotine-treated and control rats (F_((4,32))=16.62, p<0.001). There wasno difference in the effects of CGP44532 in nicotine-treated compared tocontrol rats (group×dose interaction: F_((4,32))=0.05, NS). CGP44532also had no effect on response latencies at any dose tested(F_((4,32))=0.30, NS). TABLE 1 Effects of the GABA_(B) receptor agonistCGP44532 on ICSS thresholds and response latencies in nicotine- andvehicle-treated rats Vehicle Nicotine CGP44532 Thresholds LatenciesThresholds Latencies (mg/kg) n = 5 n = 5 0  96.79 ± 2.8  95.51 ± 2.2 99.93 ± 4.1 100.31 ± 4.6  0.065  96.28 ± 3.1  97.31 ± 3.8  96.37 ± 3.2106.02 ± 8.1  0.125 106.76 ± 3.2 102.34 ± 2.8 103.88 ± 7.2 98.15 ± 5.30.25 109.14 ± 5.1 105.03 ± 3.1 109.98 ± 5.4 99.05 ± 3.1 0.5   148.43 ±16.6*** 106.13 ± 6.5   146.83 ± 14.3*** 98.11 ± 5.4Data (mean ± SEM) are expressed as percentage of baseline ICSS thresholdand response latency.Asterisks indicate statistically significant differences betweenCGP44532 and vehicle treatment.***P < 0.001 after significant two-way ANOVA with repeated measures.

As shown in FIG. 3, bilateral microinfusion of LY314582 (10-100 ng/side)directly into the VTA significantly elevated reward thresholds innicotine-treated but not control rats. Again there were significanteffects of group (F_((1,13))=4.81, p<0.05), dose (F_((3,39))=4.77,p<0.01), and a significant group×dose interaction (F_((3,39))32 3.82,p<0.05). Post-hoc analyses revealed that doses of 50 and 1 00 ng/sideLY31 4582 were sufficient to elevate reward thresholds innicotine-treated rats without affecting thresholds in control rats.LY314582 had no effect on response latencies (F_((3,39))=1.94, NS) innicotine-treated or control rats after VTA administration (FIG. 3B).

As shown in FIG. 4, withdrawal from chronic nicotine treatment producedrobust ICSS threshold elevations compared to control rats(F_((1,27))=15.3, p=0.0006). Analysis of the significant group×dose×timeinteraction (F_((5,135))=3.3, p=0.01) revealed the following:Nicotine-treated rats injected with vehicle demonstrated robust rewardthreshold elevations that reached a peak 24 h following mini-pumpremoval (FIG. 4A). However, administration of LY341495 30 min before the18 h time-point significantly attenuated the elevations in rewardthresholds in nicotine-withdrawing rats (p<0.001) (see FIG. 4A), withoutaffecting thresholds in control rats (FIG. 4B). LY341495 had no effecton response latencies at any time-point after injection(F_((1,27))=0.43, NS).

As illustrated in Table 2, MK-801 (dizocilpine) (0.01-0.2 mg/kg) loweredreward thresholds in nicotine-treated and control rats (F_((6,66))=7.5,p<0.0001). TABLE 2 Effects of the NMDA receptor antagonist MK-801 onICSS thresholds and response latencies in nicotine- and vehicle-treatedrats Vehicle Nicotine Thresholds Latencies Thresholds Latencies MK-801(mg/kg) n = 9 n = 10 0 102.72 ± 3.9    99.04 ± 1.7 103.65 ± 1.7  93.73 ±2.7 0.01 95.99 ± 3.3  100.00 ± 2.5 98.20 ± 6.1 95.41 ± 2.3 0.03 92.54 ±2.6  103.14 ± 2.1 93.09 ± 3.9 94.64 ± 3.6 0.1 88.48 ± 5.6**  98.97 ± 3.989.42 ± 3.6 106.14 ± 4.4  0.15 87.14 ± 4.6** 101.60 ± 2.0   76.24 ±4.7*** 93.65 ± 3.6 0.175 93.64 ± 3.4*    119.22 ± 11.9**  82.26 ± 3.7**93.56 ± 4.2 0.2  82.47 ± 4.3***  119.21 ± 9.4**  84.29 ± 3.0* 105.14 ±1.9 Data (mean ± SEM) are expressed as percentage of baseline ICSS thresholdand response latency.Asterisks indicate statistically significant differences between MK-801and vehicle treatment.*P < 0.05,**p < 0.01,***p < 0.001 after significant two-way ANOVA with repeated measures.

MK-801 lowered reward thresholds by a similar magnitude innicotine-treated and control rats and there was no group×doseinteraction (F_((6,66))=1.2, NS). Doses of MK-801 ≧0.2 mg/kg causeddisruption in performance in the ICSS paradigm in both groups such thatrats no longer responded for self-stimulation, and therefore doseshigher than 0.2 mg/kg were not tested. Further, MK-801 did notprecipitate withdrawal-like elevations in reward thresholds innicotine-treated rats at any dose tested. MK-801 did significantlyincrease response latencies (F_((6.72))=2.9, p<0.05). Post-hoc analysisdemonstrated that as the dose of MK-801 increased so too did responselatency, particularly in control rats, suggesting that performance wasincreasingly impaired at higher doses of MK-801.

As shown in Table 3, low doses of MPEP (0.01-0.1 mg/kg) did not affectreward thresholds (F_((3,39))=2.3, NS) or response latencies(F_((3,39))=0.4, NS) in nicotine-treated or control rats. Higher dosesof MPEP (0.5-3 mg/kg) elevated reward thresholds in nicotine-treated andcontrol rats (F_((4,96))=8.4, P<0.0001). However, MPEP elevated ICSSthresholds in both groups of rats by a similar magnitude, and there wasno group×dose interaction (F_((4,96))=0.7, NS). MPEP (0.5-3 mg/kg) hadno effect on response latencies (F_((4,9))=1.4, NS) in either group.TABLE 3 Effects of the mGlu5 receptor antagonist MPEP on ICSS thresholdsand response latencies in nicotine- and vehicle-treated rats MPEPVehicle Nicotine (mg/kg) Thresholds Latencies Thresholds Latencies n = 7n = 8 0  97.67 ± 2.0 104.78 ± 1.1  95.68 ± 3.5 101.79 ± 5.2 0.01  98.75± 3.5  99.75 ± 3.4  93.86 ± 6.0 101.50 ± 2.2 0.05 101.27 ± 4.1 104.87 ±3.3  94.11 ± 2.6 103.48 ± 4.2 0.1 105.13 ± 3.2 105.13 ± 4.8 103.84 ± 1.2102.54 ± 1.5 n = 13 n = 13 0  99.91 ± 1.8  99.41 ± 1.9 100.62 ± 3.3100.76 ± 2.3 0.5 103.01 ± 2.7 103.20 ± 4.4 104.02 ± 2.2  99.47 ± 1.9 1103.70 ± 3.0 100.72 ± 2.1 105.81 ± 3.3  99.12 ± 1.7 2  111.21 ± 2.3**107.60 ± 2.6  121.17 ± 6.2** 101.31 ± 2.2 3  111.52 ± 4.8** 103.58 ± 2.3 119.11 ± 5.5** 102.22 ± 3.6Data (mean ± SEM) are expressed as percentage of baseline ICSS thresholdand response latency.Asterisks indicate statistically significant differences between MPEPand vehicle treatment.**P < 0.01 after significant two-way ANOVA with repeated measures.

As illustrated in FIG. 5, the AMPA/Kainate receptor antagonist NBQX(0.01-1 mg/kg) significantly altered ICSS reward thresholds innicotine-treated but not control rats. This effect was reflected in astatistically significant effect of group (F_((1,20))=10.82, p<0.005), asignificant effect of dose (F_((7,140))=2.8, p<0.01), and a significantgroup×dose interaction (F_((7,140))=2.1 1, p<0.05). Post-hoc analysisrevealed a bimodal action of NBQX on reward thresholds innicotine-treated rats. Low doses of NBQX (0.025-0.1 mg/kg) elevatedreward thresholds in nicotine-treated rats, whereas higher doses of NBQX(0.5-1 mg/kg) were less effective and did not significantly increasethresholds compared to vehicle treatment (FIG. 5A). NBQX had no effecton response latencies in nicotine-treated or control rats at any dosetested (F_((7,140))=0.31, NS) (FIG. 5B).

The present data provide strong evidence for a role of Group IImetabotropic glutamate receptors in regulating the deficit in brainreward function associated with nicotine withdrawal by demonstratingthat activation of mGluII receptors precipitated reward deficits innicotine-treated rats similar to those observed during nicotinewithdrawal. Further, activation of mGluII receptors located within theVTA also precipitated withdrawal-like reward deficits in nicotinetreated rats, providing further evidence for an important role of theVTA in mediating the actions of nicotine on the brain's reward pathway(Hildebrand et al., 1999; Mansvelder and McGehee, 2000; Mansvelder etal., 2002). Finally, blockade of mGluII receptors attenuated the deficitin brain reward function observed in rats undergoing spontaneousnicotine withdrawal. Previously, the mGluII receptor agonist LY354740was shown to attenuate the increased auditory startle observed duringspontaneous nicotine withdrawal (Helton et al., 1997). One possibleexplanation for these observations is that mGluII receptors located indifferent brain sites may differentially regulate various aspects ofnicotine withdrawal.

Discussion

The present invention is based on the experimental results presented inthe Examples, related to the role and possible mechanisms of action ofmGluII and GABA_(B) receptors in mediating the reward deficitsassociated with nicotine withdrawal. Systemic administration of theselective mGluII receptor agonist LY314582 (2.5-7.5 mg/kg), but not theGABA_(B) receptor agonist CGP44532 (0.065-0.5 mg/kg), precipitatedwithdrawal-like elevations in intracranial self-stimulation (ICSS)reward thresholds, a sensitive measure of reward function, innicotine-dependent but not control rats. LY314582 did not affectresponse latencies, a measure of performance in the ICSS paradigm.Bilateral microinfusion of LY314582 (10-100 ng/side) into the ventraltegmental area (VTA) likewise precipitated threshold elevations innicotine-dependent but not control rats. Further, a single injection ofthe mGluII receptor antagonist LY341495 (1 mg/kg) attenuated theelevations in reward thresholds in rats undergoing spontaneous nicotinewithdrawal. Finally, because activation of mGluII receptors decreasesglutamate transmission, it was hypothesized that blockade ofpostsynaptic glutamate receptors would also precipitate withdrawal-likereward deficits in nicotine-dependent rats. Accordingly, NBQX (0.001-1mg/kg), a selective AMPA/Kainate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate) receptor antagonist, precipitated withdrawal-likeelevations in ICSS thresholds in nicotine-dependent but not controlrats, whereas MPEP (0.01 -3 mg/kg) and MK-801 (0.01-0.125 mg/kg),antagonists at mGlu5 (metabotropic glutamate5) and NMDA(N-methyl-D-aspartate) receptors respectively, did not. Overall, thesedata demonstrate that inhibitory regulation of brain reward function bymGluII receptors located in the VTA was increased in nicotine-dependentrats, which contributed to the reward deficits associated with nicotinewithdrawal. Further, it is likely that decreased glutamate transmissionat AMPA/Kainate receptors also contributed to nicotinewithdrawal-induced reward deficits.

Not to be limited by theory, the present invention is based in part onthe following considerations. There is now compelling evidence that theaversive syndrome observed during periods of nicotine abstinencecontributes to tobacco addiction (Kenny and Markou, 2001, PharmacolBiochem Behav 70:531-549). Indeed, nicotine withdrawal recently wasshown to precipitate a deficit in brain reward function, as measured byelevations in intracranial self-stimulation (ICSS) reward thresholds,similar in magnitude and duration to that observed in rats undergoingwithdrawal from other major drugs of abuse (Epping-Jordan et al., 1998Nature 393:76-79; Harrison et al., 2001 Neuropsychopharmacology25:55-71). Moreover, this deficit in brain reward function has beenproposed as a major motivational factor contributing to craving, relapseand continued tobacco consumption in human smokers (Epping-Jordan etal., 1998). However, in contrast to the intense investigation into themechanisms by which nicotine produces its rewarding effects (seePicciotto and Corrigall, 2002 J Neurosci 22:3338-3341), little is knownregarding the mechanisms mediating the reward deficits associated withnicotine withdrawal.

A common feature of drugs of abuse including nicotine is their abilityto increase dopamine transmission in the mesoaccumbens reward pathwayand thereby facilitate brain reward function (Pontieri et al., 1996;Picciotto, 1998; Di Chiara, 2000). Nicotine is thought to achieve this,in part, by activation of excitatory glutamate transmission in the VTA(Schilström et al., 1998; Mansvelder and McGehee, 2000; Mansvelder etal., 2002). Therefore, because mGluII receptors located in the VTA arepresynaptic autoreceptors that decrease glutamate transmission (Bonci etal., 1997; Wigmore and Lacey, 1998; Manzoni and Williams, 1999), it islikely that the increased sensitivity of mGluII receptors located in theVTA in nicotine-treated rats occurred in response to a pattern ofprolonged activation of excitatory glutamate transmission by nicotine inthis brain site.

During nicotine withdrawal, when the stimulatory effects of nicotine onexcitatory glutamate transmission were no longer present, increasedmGluII receptor function in the VTA would be expected to decreaseglutamate transmission and thereby decrease the activity of the brain'sreward system. Electrophysiological and in vivo microdialysis studiesare consistent with this hypothesis. For instance, Manzoni and Williams(1999) demonstrated that prolonged opiate treatment increased theinhibitory effects of mGluII receptor agonists on excitatory glutamatecurrents in VTA dopamine neurons. Similarly, repeated cocaine treatmentincreased the content and dimerization of mGluII receptors in the NAcc,and thereby increased the inhibitory effects of mGluII receptors onglutamate efflux in this brain site (Xi et al., 2001). It is interestingto note that similar studies also documented an increase in GABA_(B)receptor function in the VTA after repeated treatment with opiates andamphetamine (Manzoni and Williams, 1999; Giorgetti et al., 2002).However, we found that regulation of brain reward function by GABA_(B)receptors was unchanged in nicotine-treated rats compared to controls,suggesting that GABA_(B) receptors are probably not involved inmediating the reward deficits associated with nicotine withdrawal.

Previous studies have found that the mGluII receptor agonist LY354740ameliorated the increased auditory startle response in rats undergoingspontaneous nicotine withdrawal (Helton et al., 1997). Similarly,agonists at mGluII receptors decreased physical signs of abstinence inrats undergoing opiate withdrawal (Fundytus and Coderre, 1997;Vandergriff and Rasmussen, 1999). These observations may appear atvariance with the data presented here in which systemic and intra-VTAadministration of a selective mGluII receptor agonist precipitatedwithdrawal-like reward deficits in nicotine-treated rats and blockade ofthese receptors reversed spontaneous nicotine withdrawal.

However, there is accumulating evidence for a differential role ofglutamate transmission in various aspects of drug withdrawal. Forexample, increased glutamate transmission, particularly in the locuscoeruleus (LC) and amygdala (Zhang et al., 1994; Rasmussen, 1995; Tayloret al., 1997), is thought to play an important role in the expression of‘somatic’ aspects of the opiate withdrawal syndrome, whereas decreasedglutamate transmission in the mesoaccumbens reward system is thought tocontribute to the reward and motivational deficits manifest duringwithdrawal from opiates and other drugs of abuse (Keys et al., 1998; Luand Wolf, 1999; Manzoni and Williams, 1999; Giorgetti et al., 2002).Indeed, Shaw-Lutchman and colleagues (2002) recently demonstrated thatcAMP response element (CRE)-mediated transcription, a marker of cellularactivity, was increased in the LC and amygdala and decreased in the VTAin rats undergoing precipitated morphine withdrawal, suggesting that theactivity of these site was altered in opposite directions duringmorphine withdrawal. Based on these observations, it is likely that asimilar dissociation exists also for the role of glutamate in mediatingdifferent aspects of nicotine withdrawal. However, it is worth notingthat affective aspects of drug withdrawal, and in particular the deficitin brain reward function, are considered more important in themaintenance of dependence to drugs compared to other symptoms of drugwithdrawal (Markou et al., 1998; Kenny and Markou, 2001; Ahmed et al.,2002).

Glutamate transmission regulates the excitability of mesoaccumbensdopamine neurons, and plays an essential role in mediating baselineactivity of reward pathways (Kalivas and Stewart, 1991; Suaud-Chagny etal., 1992; Kalivas, 1993). As described above, mGluII receptors act aspresynaptic autoreceptors in the VTA where they depress glutamatetransmission. Therefore, it was hypothesized that mGluII receptorselevated reward thresholds in nicotine-withdrawing rats, at least inpart, by decreasing glutamate transmission and that blockade ofglutamate transmission at postsynaptic glutamate receptors wouldprecipitate reward threshold elevations in nicotine-treated rats similarto those in rats undergoing spontaneous nicotine withdrawal.

Accordingly, at low doses the AMPA/Kainate receptor antagonist NBQXprecipitated elevations in reward thresholds in nicotine-treated but notcontrol rats. This observation suggests that decreased glutamatetransmission at AMPA/Kainate receptors contributes to the rewarddeficits associated with nicotine withdrawal. These data are alsoconsistent with recent findings demonstrating that prolonged nicotinetreatment decreased AMPA receptor immunoreactivity in the NAcc and VTA(Lee et al., 2001). Under normal conditions, AMPA/Kainate receptors arethe primary regulators of excitatory glutamate transmission throughoutthe mesoaccumbens reward pathway (Pennartz et al., 1990; Hu and White,1996). Therefore, similar to an increase in mGluII receptor function,decreased AMPA receptor expression in nicotine-treated rats would beexpected to decrease mesoaccumbens dopamine transmission and thereby actto counter the prolonged stimulatory effects of nicotine on this system.

Curiously, blockade of NMDA receptors in nicotine-treated rats did notprecipitate reward threshold elevations, but instead lowered thresholds,reflecting a rewarding action in both nicotine-treated and control rats.There is considerable evidence that NMDA receptors play a crucial rolein mediating the stimulatory effects of nicotine on mesoaccumbensdopamine transmission (Schilström et al., 1998; Fu et al., 2000;Mansvelder and McGehee, 2000). Therefore, it might have been expectedthat by blocking nicotine's stimulatory effects on reward pathways byantagonizing NMDA receptors, the counter-adaptive processes thatoccurred during prolonged nicotine treatment, such as increasedsensitivity of mGluII receptors, would have given rise to elevations inICSS thresholds.

One explanation for these data is that many populations of NMDAreceptors exist throughout the brain's reward circuitries, potentiallywith opposite actions on brain reward function. Indeed, antagonists atNMDA receptors are by themselves rewarding and have been shown to lowerICSS thresholds after direct intra-VTA administration (David et al.,1998). Blockade of mGlu5 receptors also did not precipitate nicotinewithdrawal, but instead elevated ICSS thresholds in control andnicotine-treated rats. These data demonstrate that mGlu5 receptorblockade decreases brain reward function to a similar extent in bothgroups of rats.

References Cited in Example 1

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EXAMPLE 2 The mGluR5 Antagonist MPEP Decreases NicotineSelf-Administration in Rats and Mice

This example illustrates that blockade of mGluR5 decreases nicotineself-administration in both rats and mice, and are consistent withfindings showing a role of mGluR5 in cocaine self-administration.Nicotine self-administration by rats is thought to reflect the rewardingeffects of nicotine, and has been shown to be sensitive to modulation ofdopaminergic (Corrigall & Coen 1991; Picciotto and Corrigall 2002),cholinergic (Watkins et al. 1999; Corrigall et al. 2002; Picciotto andCorrigall 2002) and K-amino-butyric acid (GABA)-ergic (Dewey et al.1999; Paterson and Markou 2002; Picciotto and Corrigall 2002)neurotransmission. Additional work has suggested a possible role forglutamate in mediating the rewarding effects of nicotine (McGehee et al.1995; Schilstrom et al. 2000; Reid et al. 2000). Specifically,neurochemical studies indicated that systemic nicotine administrationresulted in significant increases in glutamate levels in the ventraltegmental area (VTA) and the nucleus accumbens (NAcc) (Reid et al. 2000;Schilstrom et al. 2000). Further, a recent electrophysiological study byMansvelder and colleagues (2002) indicated a complex interaction betweenGABA-ergic and glutamatergic inputs to midbrain dopamine neurons at thelevel of the VTA in slices exposed to nicotine levels of similarconcentration and duration as those experienced in smoker's brains. Insummary, nicotine is thought to exert its rewarding effects by complexinteractions of multiple neurotransmitters in the central nervoussystem. Chief amongst these is the mesolimbic dopaminergic system,although recently glutamate also has been shown to play an importantrole.

The eight subtypes of metabotropic glutamate receptors (mGluR) aredivided into three groups, based on amino acid sequence homology, secondmessenger systems and pharmacology (Conn and Pin 1997). The Group ImGluRs comprise metabotropic glutamate receptor subtype 5 (mGluR5) andmGluR1, and act via stimulation of phospholipase C (Conn and Piri 1997).The distribution of mGluR5s has been characterized in detail, indicatingheavy distribution in the NAcc, striatum and hippocampus in the ratbrain (Shigemoto et al. 1993; Tallakssen-Greene et al. 1998).Metabotropic glutamate receptors have been shown to be involved in themodulation of neurotransmitter release (Cartmell and Schoepp 2000;Schoepp 2001), synaptic plasticity and learning (Holscher et al. 2001),neuroprotection (Battaglia et al. 2001, 2002), dopamine-dependentlocomotor behaviors (for review, see Vezina and Kim 1999) and pain (forreview, see Spooren et al. 2001).

Recently, and most relevant to the present study, it has been shown thatmice lacking mGluR5 will not acquire intravenous cocaineself-administration (Chiamulera et al. 2001). In addition,administration of the specific mGluR5 antagonist2-methyl-6-(phenylethynyl)-pyridine (MPEP; Gasparini et al. 1999)resulted in a dose-dependent reduction in cocaine self-administrationwithout affecting responding for food (Chiamulera et al. 2001).Furthermore, it has been shown that repeated cocaine administration overone week resulted in significantly increased levels of mGluR5 mRNAexpression in the nucleus accumbens shell and dorsal striatum thatpersisted for three weeks after the cessation of cocaine administration(Ghasemzadeh et al. 1999). In addition to cocaine self-administration,MPEP has been shown to have several effects in a variety of behavioralparadigms in laboratory animals. Previous work indicated thatadministration of MPEP in rodents has significant anxiolytic- andantidepressant-like effects (Spooren et al. 2000b; Tatarczynska et al.2001). In addition, MPEP was shown to block fear conditioning (Schulz etal. 2001), exert anti-Parkinsonian-like effects (Ossowska et al. 2001;Breysse et al. 2002), and have anticonvulsant activity (Chapman et al.2000) in rodents. In summary, MPEP has been shown to have a wide varietyof effects in a number of different behavioral paradigms in rodents, andthe mGluR5 has been shown to be involved in cocaine self-administrationand possibly longer-term effects of cocaine.

Based on previous findings summarized above indicating a role of mGluR5receptors in mediating the rewarding effects of cocaine (Chiamulera etal., 2001), and also the role of glutamate in mediating the rewardingeffects of nicotine, the present study set out to determine the effectsof MPEP on the reinforcing effects of intravenous nicotine in rats thatregularly self-administer nicotine at one of two doses (0.03 and 0.01mg/kg/infusion), and in drug-naive mice. In addition, the effect of MPEPadministration on food-maintained responding in rats was investigated todetermine the specificity of the effects of MPEP. Further, the mouseself-administration procedure used here includes yoked control mice thatallow the assessment of non-specific effects of pharmacologicalmanipulations.

Materials and Methods

Subjects

Male Wistar rats (Charles River, Raleigh, N.C.) weighing 300-350 g uponarrival in the laboratory were group housed (two per cage) in atemperature- and humidity-controlled vivarium on a 12 hr reverselight-dark cycle (lights on at 10 am) with unrestricted access to waterexcept during testing, and food-restricted to 20 g/day after acquiringnicotine self-administration behavior. Male DBA/2J mice (HarlanLaboratories, Indianapolis, Ind.) at the age of 10-12 weeks upon arrivalin the laboratory were group housed (4 per cage) in a temperature- andhumidity-controlled vivarium on a 12 hr reverse light-dark cycle (lightson at 6 am) with unrestricted access to food and water except duringtesting. All behavioral testing occurred during the dark phase of thelight-dark cycle. All subjects were treated, housed and used inaccordance with the National Institutes of Health guidelines and theAssociation for the Assessment and Accreditation of Laboratory AnimalCare (AAALAC).

Drugs

(−)Nicotine hydrogen tartrate was purchased from Sigma (St. Louis, Mo.),dissolved in saline and the pH adjusted to 7.0 (±0.5) with sodiumhydroxide. The solution was then filtered through a 0.22 μm syringefilter (Fisher Scientific, Pittsburgh, Pa. 15219) for sterilizationpurposes. Nicotine doses are reported as free base concentrations. MPEPwas kindly donated by Novartis Pharma AG, dissolved in 0.9% sodiumchloride, and administered intraperitoneally in a volume of 1 or 10ml/kg with a pretreatment time of 30 or 15 minutes in rats and mice,respectively.

Apparati

Rat Self-Administration and Food Responding Operant Boxes

Intravenous nicotine self-administration and food-maintained respondingtook place in twelve Plexiglas operant chambers each housed in asound-attenuated box as described previously (Markou and Paterson 2001).

Mouse Self-Administration Operant Boxes

All nicotine self-administration in mice took place in an apparatus (SanDiego Instruments, San Diego, Calif.) that consisted of four identicaltest cages (8×8×8 cm), allowing simultaneous testing of two pairs ofmice (see Semenova et al. 1995, 1999; Kuzmin et al. 1996a, 1997). Theboxes were constructed form opaque plastic and contained two apertures:one for nose-pokes, and one for tail-fixing. All test session functionsand data were controlled and recorded by computer.

Rat Self-Administration and Food-Maintained Responding

Food Training and Acquisition of Nicotine Self-Administration

Approximately one week after preparation with catheters into the jugularvein (see Markou and Paterson 2001), rats were trained toself-administer nicotine (see Markou and Paterson 2001) at two doses(0.01 [n=9] and 0.03 [n=9] mg/kg/inf; free base) under a FR5 TO20 secschedule. Two additional groups of rats were allowed to respond for food(45 mg Noyes food pellet) under a FR5 TO20 sec (n=10), and a FR5 TO210sec (n=10) schedule. The two different schedules were employed to allowassessment of the effects of MPEP on responding for food under areinforcement schedule (FR5 TO20 sec) identical to that used fornicotine, and under a reinforcement schedule (FR5 TO210) that equatedresponse rates for food to those seen for nicotine. Responding on theactive lever resulted in delivery of nicotine solution in a volume of0.1 ml/infusion over a 1 second period (Razel Scientific InstrumentsInc, Stamford, Conn.), while responding on the inactive lever had noconsequences. Rats were considered to have acquired stable operantresponding when they pressed the active lever more than twice the numberof times they pressed the inactive lever, received a minimum of 6infusions or 90 pellets per 1 hour session, with less than 20% variationin the number of reinforcers earned per session. Rats took approximatelytwo weeks to establish stable rates of operant responding. All testsessions were conducted for one hour per day, five days per week.

Intravenous Self-Administration in Mice

Acute self-administration of nicotine was carried out using a previouslydescribed method (Semenova et al. 1995, 1999; Kuzmin et al. 1996a,1997). The tails of the mice were fixed to the surface of the apparatusduring the testing sessions to allow drug delivery; the fixing of thetail allowed the mice to move all of their four paws, head and entirebody. Infusions of drug or vehicle (1.6 μl/inf delivered over 1 second)were administered to both mice in a pair, via the lateral tail-veins,contingent upon each nose-poke of one animal per pair (the activemouse). The only cue associated with the delivery of the drug or vehicleinfusion was the noise of the pump. Mice were tested either once ortwice, with approximately one month between each test. The second testonly occurred in a subset of the original set of mice (2-7 pairs of micefrom each of the original groups). This was done to increase the numberof subjects/condition, and involved randomly assigning and testing thesemice under a different set of conditions than in the original test(i.e., different nicotine and/or MPEP dose). Given the one monthinterval between the two tests for a limited subset of the mice, it isvery unlikely that repeated testing affected the results.

Experimental Procedures

Experiment 1: Effect of MPEP Administration on Rates of NicotineSelf-Administration and Food-Maintained Responding on a Fixed RatioSchedule of Reinforcement in Rats

After acquiring stable nicotine self-administration at either 0.01mg/kg/infusion (n=9) or 0.03 mg/kg/infusion (n=9), or stablefood-maintained responding on the FR5 TO 20 sec schedule (n=10) or theFR5 TO210 sec schedule (n=10), drug testing was initiated. MPEP (0, 1, 3and 9 mg/kg) was administered intraperitoneally 30 minutes before thesession, according to a Latin Square design, with at least six dayselapsing between each test dose. Drug was administered only when theanimals had demonstrated stable self-administration behavior during thepreceding 3 days, defined as less than 20% variation in dailyperformance.

Experiment 2: Effect of MPEP Administration on NicotineSelf-Administration in Drug-Naive Mice

Saline or one of four nicotine doses (0.016, 0.048, 0.16, 0.48 μg/inf)were made available to different groups of animals. Each group consistedof 9-17 pairs of animals, including 2-7 mice per group that hadpreviously undergone one testing session at least one month previously(see above). Pre-treatment with MPEP (0, 5, 10, 20 mg/kgintraperitoneally) occurred 15 minutes prior to the initiation of thetest session.

Data Analyses

Experiment 1

Data were analyzed using a two-way ANOVA, with MPEP dose as thewithin-subjects factor (4 levels) and reinforcer as the between-subjectsfactor (4 levels: 0.01 and 0.03 mg/kg/inf nicotine, and food pellets,available on the FR5 TO20 sec and FR5 TO210 sec schedules). Data wereexpressed as a percentage of baseline (defined as the mean of thepreceding three days; active lever data) and also as the number of leverpresses (inactive lever data only). The level of significance was set atp<0.05.

Experiment 2

Data analyses were based on the comparison of both active and passivemouse nose-pokes in each pair, using the formula:R=log(A_(T)/P_(T))−log(A_(BL)/P_(BL)), where (A_(T)/P_(T)) is the ratioof the total number of active versus passive mouse nose-pokes during the30-min test, and (A_(BL)/P_(BL)) is the ratio of the total number ofactive versus passive mouse nose-pokes during the 10-min pre-test. Theeffect of the drug was considered as reinforcing, neutral or aversivewhen R was higher, equal or lower than zero, respectively. Somedata-points (9/237 pairs of mice) were excluded from the final analysesbased on whether or not they lay within 2 standard deviations of thegroup mean for that specific condition. Nicotine self-administrationdata, obtained after saline pre-treatment, were analyzed using a one-wayANOVA with nicotine (4 levels) defined as the between-subjects factor.R-criterion data were analyzed using two-way ANOVAs with MPEP dose (4levels) and nicotine dose (4 levels) defined as the between-subjectsfactors. Pre-planned R-criterion data analyses were performed usingone-way ANOVAs for each available dose of self-administered nicotine andsaline, with MPEP dose (4 levels) defined as the between-subject factor.Appropriate individual comparisons were performed using theStudent-Newman-Keuls post hoc test. The total self-injected doses of0.048 μg/inf nicotine were analyzed using a one-way ANOVA with MPEP doseas the between-subjects factor, since analyses indicated that 0.048μg/inf nicotine was the only dose reliably self-administered by the mice(see Results). Further, the raw nose-poke response rates for thenicotine dose that was reliably self-administered (0.048 μg/inf) werealso subjected to a two-way ANOVA analysis with active/passive mouse asone factor, and MPEP dose as the second factor. Body weights andpre-test nose-poke activity levels were analyzed with three-way ANOVAs(nicotine dose, MPEP dose and mouse, i.e. active/passive, defined asbetween-subjects factors).

Results

Experiment 1: Effect of MPEP Administration on Rates of NicotineSelf-Administration and Food-Maintained Responding on a Fixed RatioSchedule of Reinforcement in Rats

A significant MPEP×Reinforcer interaction effect [F(9,102)=4.22,p<0.001] indicated that MPEP affected nicotine self-administration andfood-maintained responding differently (FIG. 6; Table 4). Further, therewere significant main effects of MPEP dose (F[3,102]=23.33, p<0.001) andreinforcer (F[3,34]=14.07, p<0.001) (FIG. 7). Newman-Keuls post-hoctests indicated that 3 and 9 mg/kg MPEP significantly reducedself-administration of the 0.01 mg/kg/infusion nicotine dose relative tothe vehicle condition, while decreasing self-administration of the 0.03mg/kg/inf dose only at 9 mg/kg. In contrast to the nicotine-maintainedresponding, food-maintained responding under either the FR5 TO20 sec orthe FR5 TO210 sec schedules was not significantly reduced at any MPEPdose administered. Analysis of inactive lever data indicated that therewas no effect of MPEP on inactive lever presses for any of thereinforcers, as indicated by the lack of a main effect of MPEP dose.There was an effect of reinforcer on inactive lever presses[F(3,34)=4.47, p<0.01], but there was no significant MPEP×Reinforcerinteraction. TABLE 4 Number of reinforcers earned during rat nicotineself-administration and food-maintained responding during the entire 1hour session. The data are expressed as the range of raw values seen inall experimental subjects under baseline conditions, and also as themean ± SEM of reinforcers earned after MPEP (0, 1, 3 and 9 mg/kg)pretreatment. 0 mg/kg 1 mg/kg 3 mg/kg 9 mg/kg Reinforcer Schedule MPEPMPEP MPEP MPEP 0.01 mg/kg/inf FR5 TO20 sec 18.9 ± 2.8 21 ± 3.1   11 ±2.7 9.1 ± 3.0 nic 0.03 mg/kg/inf FR5 TO20 sec 12.9 ± 1.2 13 ± 1.9 11.4 ±1.2 4.8 ± 1.5 nic 45 mg food pellet FR5 TO20 sec 122.7 ± 6.0  131.3 ±8.2   122.2 ± 7.4    109 ± 12.98 45 mg food pellet FR5 TO210 sec 16.1 ±0.4 16.3 ± 0.3   15.2 ± 0.5 15.2 ± 0.6 

Experiment 2: Effect of MPEP Administration on NicotineSelf-Administration in Drug-Naive Mice

Statistical analysis of R-criterion data indicated that drug-naive miceacquired nicotine self-administration as indicated by a significant maineffect of nicotine [F(4,71)=3.4, p<0.05]. Post-hoc comparisons indicatedthat self-administration of the 0.048 μg/inf nicotine dose wassignificantly different from saline administration (FIG. 8). Statisticalanalyses of R-criterion data indicated there was a trend towards asignificant interaction of MPEP×Nicotine dose [F(3,90=1.89, p=0.056].Based on pre-planned comparisons for the effect of MPEP onself-administration of each nicotine dose, one-way ANOVAs indicated thatMPEP had a significant effect only on self-administration of the 0.048μg/inf nicotine dose [F(3,46)=3.53, p<0.05], that was actually the onlynicotine dose to be reliably self-administered. Post-hoc comparisonsindicated that MPEP significantly reduced nicotine self-administrationat all doses tested (FIG. 8). Interestingly, FIG. 8A appears to show atrend for 20 mg/kg MPEP to decrease the R-criterion below zero, perhapsindicating that this dose of MPEP makes nicotine aversive, under thetest conditions. Analyses indicated no significant effect of MPEP ontotal self-injected nicotine dose (FIG. 8B). Nevertheless, MPEPsignificantly decreased nose-poke responding in the active mice, asindicated by a Mouse (Active vs Passive)×MPEP interaction effect[F(3,96)=3.46, p<0.05)]. There was no difference between any of theexperimental groups for pre-test nose-poke activity. In addition, therewere no significant differences in body weights between the differentconditions, as indicated by a lack of a significant three-wayinteraction. Table 5 shows the raw data for each group of mice,expressed as the number of nose-pokes per minute, recorded during thepre-test and during the self-administration session, for both active andpassive mice. TABLE 5 Raw data from the mouse self-administrationexperiment. The data in the table include the sample size for eachcondition, expressed as the number of pairs of mice, and also rates ofnose-pokes responses recorded during both pre-test and test sessions,under all conditions, for both active and passive subjects, expressed asmean responses per minute ± SEM. For the 0.048 μg/kg/inf nicotine dose,there was a significant interaction between the factors mouse (active vspassive) and the factor MPEP indicating that MPEP differentiallyaffected nose-poke rates in the active versus the passive mice. MPEPpassive pre- Nicotine (μg/inf) (mg/kg) n active pre-test active testtest passive test 0 0 15 8.79 ± 0.98 4.62 ± 1.05 9.37 ± 1.11 4.05 ± 0.820 5 10 8.19 ± 1.03 4.99 ± 1.17 8.38 ± 0.96 6.41 ± 1.38 0 10 10 7.95 ±0.99 4.92 ± 1.1  8.00 ± 1.06 5.86 ± 1.56 0 20 10 6.98 ± 1.18 5.96 ± 1.698.50 ± 1.43 6.33 ± 1.66 0.016 0 13  8.0 ± 1.22 3.88 ± 0.46 8.85 ± 1.254.56 ± 0.86 0.016 5 11 7.86 ± 0.87 2.62 ± 0.49 8.01 ± 0.91 3.46 ± 0.740.016 10 13 7.79 ± 0.83 6.48 ± 1.05 8.22 ± 0.87 4.26 ± 0.94 0.016 20 107.10 ± 0.87 5.48 ± 1.09 7.35 ± 0.85 8.77 ± 1.85 0.048 0 17 8.04 ± 0.895.42 ± 1.19 8.50 ± 1.07 3.06 ± 0.68 0.048 5 12 9.13 ± 1.37 3.43 ± 0.678.93 ± 1.36 4.45 ± 1.06 0.048 10 11 7.43 ± 0.98 3.92 ± 1.18 8.06 ± 1.395.83 ± 1.72 0.048 20 10 8.36 ± 1.07 3.56 ± 1.04 8.31 ± 1.28 5.29 ± 1.110.16 0 16 7.58 ± 0.77 3.63 ± 0.58 7.69 ± 0.88  3.9 ± 0.69 0.16 5 14 8.32± 0.94 4.67 ± 0.95 8.48 ± 3.31 4.32 ± 2.37 0.16 10 11 7.12 ± 0.66 4.19 ±0.81 7.20 ± 0.69 4.62 ± 0.82 0.16 20 10 7.50 ± 1.25 5.06 ± 1.52 7.56 ±1.32 7.97 ± 1.76 0.48 0 15 6.50 ± 0.90 2.29 ± 0.25 6.77 ± 0.87 3.28 ±0.5  0.48 5 9 8.12 ± 1.17 3.04 ± 0.73 8.58 ± 1.38 3.25 ± 0.88 0.48 10 107.49 ± 0.86 3.58 ± 0.73 7.48 ± 0.88 3.81 ± 0.47 0.48 20 10 7.31 ± 0.984.67 ± 1.22 7.08 ± 0.94 4.35 ± 0.98

Discussion

The results of the present study indicated that MPEP administrationselectively decreased nicotine self-administration in the rat withincreased efficacy at the lower available nicotine dose, withoutaffecting food-maintained responding on either of the twofood-maintained schedules of reinforcement employed. In addition, MPEPadministration in drug-naive mice was shown to suppressself-administration of the nicotine dose that was shown to be reliablyself-administered (0.048 μg/inf nicotine). These data indicate that MPEPpretreatment reduced the reinforcing efficacy of self-administeredintravenous nicotine in two different rodent species.

In the rats there were no obvious motor suppressant effects that couldaccount for the reduction in nicotine self-administration, as indicatedby the maintenance of high levels of responding for food on either ofthe two schedules of reinforcement, one of which equated response ratesto those seen for nicotine. In the mice experiment there was no effectof MPEP administration on nose-poke activity. Previous studies generallyhave indicated no effects of MPEP on locomotor activity in rodents. Inrats, MPEP at doses of 2.5 -10 mg/kg, delivered intraperitoneally(i.p.), had no effect on exploratory locomotor activity in rats(Tatarczynska et al. 2001; Henry et al. 2002), although another studyindicated that much higher doses of MPEP inhibited spontaneous locomotoractivity; but even very high doses were without effect on rotarodperformance (Spooren et al. 2000a). Similarly in mice, Tatarczynska andco-workers (2001) demonstrated that 30 mg/kg MPEP (i.p.) had no effecton rotarod performance, but a different study (Spooren et al. 2000a)showed that a much higher dose of MPEP reduced vertical activity. Insummary, previous work and the present data on food-maintainedresponding in rats indicate that at the doses of MPEP used in thepresent study, non-specific motor effects are very unlikely to accountfor the reduced nicotine self-administration. Furthermore, the observeddifferences in the effects of MPEP on nicotine-versus food-maintainedresponding cannot be attributed to a rate-dependent effect, since theuse of the FR5 TO210 sec schedule provided equivalent rates ofresponding for nicotine and food and even under these conditions MPEPdid not affect responding for food.

With the mouse self-administration technique used in the present study,mice demonstrated self-administration at one of the five doses testedacross a wide range of nicotine doses. This procedure forself-administration in drug-naive mice has been used to study theself-administration of morphine (Semenova et al. 1995, 1999; Kuzmin etal. 1996a, 1997), cocaine (Kuzmin et al. 1996b; 1997), ethanol (Kuzminet al., 1999), nicotine (Stolerman et al. 1999; Fattore et al. 2002),amphetamine (Cossu et al., 2001) and gamma-hydroxybutyric acid(Martellota et al. 1998). A wide range of nicotine doses was used in thepresent study because of uncertainty over the likely self-administerednicotine dose range in the DBA/2J mice used, and to allow for largeshifts in the nicotine dose-response curve after MPEP pretreatment.Although only limited conclusions can be drawn from studies utilizingonly one unit dose of the self-administered drug, the present mouse dataare presented in the context of, and in addition to, the extensiveself-administration studies in the rat. The partial restraint used inthe mouse procedure could increase drug self-administration behavior(Piazza et al. 1990; Shaham et al. 1993; Ramsey and Van Rees 1993),although physical stress (i.e. foot shock) was shown previously not toaffect self-administration of morphine using the present procedure(Kuzmin et al., 1996). In contrast to the mouse technique, themethodologies employed in the rat experiments in the present studyexamined self-administration behavior at two different unit doses ofnicotine, and examined the maintenance rather than acquisition ofnicotine self-administration. Furthermore, the rat studies included twofood-maintained schedules of reinforcement, with one schedule (FR5 TO20sec) identical to the schedule used for the nicotine self-administrationwork, and another schedule (FR5 TO 210 sec) used to equilibrate levelsof responding for nicotine and food. Regarding the results obtained fromthe mouse experiment, MPEP was shown to significantly reduceself-administration of the 0.048 μg/inf nicotine dose, as measured bythe R criterion. In contrast, although there was a trend towards aneffect of MPEP on total nicotine intake, it was not significant.Nevertheless, the two-way ANOVA on the raw nose-poke data at the 0.048μg/inf nicotine dose clearly indicated a differential effect of MPEPpretreatment on active versus passive mice, with the active miceexhibiting decreased nose-poke rates after MPEP administration. Insummary, although the data obtained from the mouse experiment in thepresent study, considered in isolation, provide only limited evidencefor the effect of MPEP on nicotine self-administration, they do provideconverging evidence for the more complete and persuasive data providedin the rat experiments.

The effects of MPEP on nicotine self-administration in rodents areconsistent with those seen previously for cocaine self-administration inmice (Chiamulera et al. 2001). It was shown that mutant mice with anmGluR5 deletion would not acquire cocaine self-administration at any ofthe doses effective in the wild type controls, but food-maintainedresponding was unaffected. The effect of the mGluR5 deletion wasconfirmed via administration of MPEP in wild type mice, which was shownto dose-dependently reduce cocaine self-administration without affectingfood-maintained responding. Interestingly, the authors demonstrated thatcocaine administration resulted in similar increases in dopamine levelsin the NAcc in both the mutant and wild type mice, perhaps indicatingthat increased midbrain dopamine levels alone are not sufficient tomaintain cocaine self-administration. This hypothesis is supported byrecent findings indicating that dopamine (D2) receptor knockout miceself-administer cocaine (Caine et al. 2002), and dopamine (D1) receptorknockouts acquire cocaine-induced conditioned place preference (Miner etal. 1995). Nevertheless, although there was no difference in theabsolute level of NAcc dopamine levels between the mGluR5 knockoutcompared to the wild type mice, there was an apparent difference in thetime taken for NAcc dopamine levels to increase to those levels. Perhapsit is this difference that resulted in the observed differences incocaine self-administration and cocaine-induced hyperlocomotion betweenthe wild type and the homozygous mGluR5-deficicent mutant mice.

Regarding the rewarding effects of acute nicotine, a recent study(Harrison et al. 2002) investigated the effect of MPEP administration onnicotine reward as measured in a brain reward stimulation procedure.MPEP administration had no effect on the reward-lowering effect of 0.25mg/kg nicotine (Harrison et al. 2002), although MPEP alone elevatedbrain reward thresholds. This discrepancy in results may be due to themuch higher doses (0.125 and 0.25 mg/kg i.p.) of nicotine administeredin the previous study (by the experimenter), compared to the effect ofself-administered 0.03 or 0.01 mg/kg/inf nicotine, delivered repeatedlyand intravenously. Importantly, the neurobiological substratesunderlying lateral hypothalamic self-stimulation and intravenousnicotine self-administration may be quite different (Harrison et al.2002), and it may be these differences that underlie the differenteffects of MPEP in the two studies.

A number of studies have indicated a role for mGluRs in modulatingdopamine neurotransmission in the striatum and NAcc, and generally,group I mGluRs (mGluR5 and mGluR1) are thought to be involved inmodulation of post-synaptic responses (Schoepp 2001). Interestingly, itwas found recently that intra-accumbens administration of a group ImGluR antagonist blocked the locomotor-activating effect of a D-1receptor agonist (David and Abraini 2001). As stated above, it has beenshown previously that mGluR5s are concentrated heavily in the NAcc,where significant increases in extracellular dopamine levels are seenafter administration of nicotine. In addition, the absence of mGluR5s inmice resulted in the absence of cocaine self-administration andcocaine-induced hyperactivity, and yet cocaine-induced increases in NAccdopamine were relatively unchanged (Chiamulera et al. 2001). Takentogether with the present findings indicating the effect of MPEP onnicotine self-administration, one explanation is that mGluR5s play amodulatory role in the postsynaptic response to drug-induced dopaminerelease in the NAcc, thereby decreasing nicotine self-administration.Alternatively, given the role of glutamate in mediating the rewardingeffects of both nicotine (Watkins et al. 2000) and cocaine (Zhang et al.2001), it may be that dopamine plays a less critical role in mediatingthe rewarding effects of psychostimulants than is often hypothesized.

The anxiolytic-like effect of MPEP may be related to its effects onself-administration of nicotine and cocaine. Nicotine can be anxiogenicat higher doses (File et al. 1998; Irvine et al. 2001), although it alsohas been shown to be anxiolytic at lower doses (File et al. 1998;Szyndler et al. 2001). Administration of amphetamine, cocaine andnicotine all increase plasma levels of stress hormones in laboratoryanimals (for review, see Sarnyai et al. 2001). It has been hypothesizedthat the anxiogenic effects of chronic self-administered nicotineobserved in rats may play a role in maintaining nicotineself-administration (Irvine et al. 2001). Pretreatment with anxiolyticbenzodiazepine compounds selectively reduced cocaine-maintainedresponding without affecting food-maintained behavior in rats (Goederset al. 1989; 1993; Goeders 1997). Previous studies have indicated theanxiolytic-like effects of MPEP in rodents (Spooren et al. 2000b;Tatarczynska et al. 2001). It is possible therefore that the anxiolyticeffects of MPEP may block the mildly heightened anxiety levels inducedby nicotine, and thus may block part of its rewarding effects.

In summary, the present study indicated that MPEP pretreatmentsignificantly reduced nicotine self-administration with no effect onfood-maintained responding in rats and in drug-naive mice in the absenceof any motor-suppressant activity. It is hypothesized here that MPEPadministration modulated the postsynaptic response to nicotine-inducedincreased dopamine levels in the terminal field of the mesolimbiccircuit. Nevertheless, given the limits of the present experiments andcurrent knowledge on the precise location and function of mGluR5s, modesof action of MPEP at sites other than the NAcc and/or via ananxiolytic-like effect, cannot be ruled out. The present results mayindicate a novel therapeutic target for anti-smoking, and moregenerally, anti-abuse medications.

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EXAMPLE 3 Supplemental Evidence for the Utility of Simultaneous Blockadeof Metabotropic Glutamate 5 and Metabotropic Glutamate 2/3 Receptors inthe Treatment of Drug Addiction and Depression

This Example illustrates that blockade of glutamatergic transmission atmGlu5 and/or mGlu2/3 receptors decreases the reinforcing properties ofcocaine and nicotine (present data; Paterson et al. 2003), and indicatesthat simultaneous blockade of these receptors has additive effects oninhibiting drug-taking behavior. Most drugs of abuse have been shown toincrease excitatory glutamatergic transmission throughout brain rewardcircuitries (Kalivas and Duffy, 1998; Mansvelder and McGehee, 2000;Ungless et al., 2001; Wolf, 2003). Although the precise role ofexcitatory glutamatergic transmission in regulating brain rewardcircuitries is unclear, increases in excitatory glutamatergictransmission contribute to the reinforcing properties of addictivedrugs. Indeed, blockade of glutamatergic transmission has been shown todecrease the rewarding actions of cocaine and other drugs of abuse(Harris and Aston-Jones, 2003; Laviolette and van der Kooy, 2003).

There has been a recent interest in the role of the metabotropicglutamate 5 (mGlu5) receptor in regulating the reinforcing effects ofaddictive drugs based on the observations of Chiamulera and colleagues(2001) demonstrating that mice in which the gene for the mGlu5 receptorwas deleted did not acquire cocaine self-administration behavior.However, the acquisition of food self-administration was unaffected inthese mice, demonstrating that the decrease in cocaine consumption inthese mice was not secondary to a deficit in learning processes(Chiamulera et al., 2001). Consistent with the above, the selectivemGlu5 receptor antagonist MPEP decreased cocaine self-administration inwild-type control mice (Chiamulera et al., 2001). Similarly, as shown inExample 2 above, MPEP decreased nicotine self-administration in bothrats and mice, and attenuated cocaine- and morphine-induced conditionedplace preference (McGeehan and Olive, 2003; Popik and Wrobel, 2002).More recently, blockade of metabotropic glutamate 2/3 (mGlu2/3)receptors has been shown to attenuate the behavioral actions of manydrugs of abuse (David and Abrain, 2003). In addition, as illustrated inExample 1, the mGlu2/3 receptor antagonist LY341495 attenuated thedeficits in brain reward function, measured by elevations in ICSS rewardthresholds, observed in rats undergoing spontaneous nicotine withdrawal(depression-like signs). Taken together, these data suggest that mGlu5and mGlu2/3 receptors play a crucial role in regulating the behavioralactions of various drugs of abuse including cocaine and nicotine, anddepressive symptoms seen in both drug addiction and non-drug-induceddepressions.

Acute cocaine administration has been shown to lower intracranialself-stimulation (ICSS) thresholds (Esposito et al., 1978; Frank et al.,1988; Kenny et al., 2003a; Kenny et al., 2003b; Kokkinidis and McCarter,1990; Markou and Koob, 1992). Because ICSS directly activates brainreward circuitries (Olds and Millner, 1954), ICSS thresholds are thoughtto provide an operational measure of brain reward function. Thus, thelowering in ICSS thresholds observed after cocaine administrationreflects an increase in brain reward function that most likely underliescocaine's euphorigenic effects. This increase in brain reward functionassociated with cocaine consumption is considered relevant to theestablishment and maintenance of cocaine self-administration behavior(Kenny et al., 2003b; Stewart et al., 1984).

The first aim of the present studies was to investigate the effects ofMPEP on nicotine self-administration in rats, and on cocaineself-administration in rats with two different schedules of daily access(1 and 6 h) to cocaine self-administration. Next, we investigatedwhether MPEP decreased the ‘motivation’ of rats to obtain cocaine ornicotine by examining whether MPEP decreased responding for cocaine ornicotine under a progressive ratio schedule of reinforcement. We theninvestigated whether MPEP decreased the positive affective stateassociated with cocaine consumption, by examining if MPEP attenuated thelowering actions of acutely administered cocaine on ICSS thresholds.Next, we examined the effects of the mGlu2/3 receptor antagonistLY341495, previously shown to attenuate nicotine withdrawal (Kenny etal., 2003c), on nicotine self-administration in rats. Finally, weexamined whether simultaneous blockade of mGlu5 and mGlu2/3 receptors,by combining MPEP and LY341495, had an additive inhibitory effect onnicotine self-administration behavior.

Materials and Methods

(i) Subjects

Subjects were Wistar rats weighing 300-320 g upon arrival at thelaboratory. Rats were obtained from Charles River Laboratories (Raleigh,N.C.) and were housed in groups of two or three per cage, with food andwater available ad libitum. Animals were maintained in atemperature-controlled vivarium under a 12 hr light/dark cycle (lightsoff at 10:00 am). In each case animals were tested during the darkportion of the light/dark cycle. All animals were treated in accordancewith the guidelines of the National Institutes of Health regarding theprinciples of animal care. Animal facilities and experimental protocolswere in accordance with the Association for the Assessment andAccreditation of Laboratory Animal Care.

Drugs

Cocaine hydrochloride and (−) nicotine hydrogen tartrate were purchasedfrom Sigma Chemical Co., St. Louis, Mo.2-Methyl-6-[phenylethynyl]-pyridine) was synthesized according toprocedures known in the art. LY341495 was obtained from Tocris. Drugswere prepared immediately before each administration. For systemiccocaine administration, cocaine was dissolved in sterile 0.9 % (w/v)saline, and administered by intraperitoneal (i.p.) injection in a volumeof 1 ml/kg body weight, 10 min before the ICSS experimental session. Forsystemic MPEP administration, MPEP was dissolved in sterile water andadministered by i.p. injection, in a volume of 1 ml/kg body weight, 30min before the ICSS or self-administration session. For systemicLY341495 administration, LY341495 was dissolved in sterile saline andadministered by i.p. injection, in a volume of 3 ml/kg body weight, 30min before the self-administration session.

Apparatus

Intracranial self-stimulation training and testing took place in sixteenPlexiglas operant chambers (25×31×24 cm) (Med Associates, St. Albans,Vt.). The floors of the operant chambers were constructed of parallelaluminum rods spaced 1.25 cm apart. One wall contained a metal wheelmanipulandum that required 0.2 N force to rotate it one-quarter of aturn. The wheel (5 cm in width) extended out of the wall ˜3 cm. Eachtesting chamber was enclosed within a light- and sound-attenuatedchamber (62×63×43 cm). Intracranial stimulation was delivered byconstant current stimulators (Stimtech model 1200; San DiegoInstruments, San Diego, Calif.). Subjects were connected to thestimulation circuit through flexible bipolar leads (Plastics One,Roanoke, Va.) attached to gold-contact swivel commutators mounted abovethe chamber. The stimulation parameters, data collection, and all testsession functions were controlled by a microcomputer.

Cocaine and nicotine self-administration took place in 16 Plexiglas,sound-attenuated operant chambers (29×24×19.5 cm). In each chamber, onewall contained a metal retractable lever that was mounted 2.5 cm abovethe floor and required a 0.1 N force to be pressed. Plastic swivels,connected the animals to syringes operated by Razel pumps that deliveredthe drug. Data collection and all programming functions were controlledby an IBM-compatible microcomputer.

Surgery

Rats prepared with intravenous catheters were anaesthetized byinhalation of 1-3% isoflurane in oxygen and prepared with silasticcatheters, surgically implanted in the jugular vein as describedpreviously (Caine et al., 1993). The catheter was passed subcutaneouslyto a polyethylene assembly mounted on the animal's back. This assemblyconsisted of a guide cannula (Plastic One Co., Roanoke Va.) attached toa 4 cm² piece of marlex mesh with epoxy. The marlex mesh was placedunder the skin on the animal's back. After surgery, catheters wereflushed daily with 0.15 ml of a sterile antibiotic solution containingheparinized saline (30 USP units/ml) and Timentin (100 mg/ml; SmithKlineBeecham Pharmaceuticals, Philadelphia, Pa.). Rats prepared with ICSSelectrodes were anaesthetized by inhalation of 1-3% isoflurane in oxygenand positioned in a stereotaxic frame (Kopf Instruments, Tujunga,Calif.). The incisor bar was adjusted to 5 mm above the interaural line,and the skull exposed. Stainless steel bipolar stimulating electrodes(11 mm in length) were implanted into the posterior lateral hypothalamus(AP: −0.5 mm from bregma; ML: ±1.7 mm; DV: 8.3 mm from dura, with theincisor bar positioned 5 mm above the interaural line), according to theatlas of Pellegrino et al. (1979). Four indentations were made in theskull to accommodate screws that, together with the application ofdental acrylic, held the electrode in place. Animals were allowed torecover from surgery for at least 7 days prior to training in the ICSSor self-administration procedures.

Intravenous Cocaine Self-Administration Procedure

Rats (n=14) were food restricted to maintain them at 85% of their normalbody weight obtained under free-feeding conditions, then trained topress a lever for 45 mg food pellets on a fixed-ratio 5 (FR5) scheduleof reinforcement. Once stable responding for food reinforcement wasachieved, rats were tested for cocaine self-administration during daily1 h sessions for nine days on a FR5 schedule of reinforcement, when fiveresponses on the lever resulted in the delivery of one cocaine injection(250 μg/injection dissolved in 0.1 ml of sterile 0.9 % sterile saline;delivered over 4 sec) and initiated a 20 sec time-out (TO) period,signaled by a light cue located above the lever, during which timeresponding on the lever was without consequence. Thus, a FR5 TO20 secschedule of reinforcement was used.

Intravenous Nicotine Self-Administration Procedure

Approximately 1 week after preparation with catheters into the jugularvein, rats (n=8) were trained to self-administer nicotine (0.03 mg/kgper infusion, free base) under an FR5 TO20 sec schedule, similar to thatdescribed above for cocaine. Responding on the active lever resulted indelivery of nicotine solution in a volume of 0.1 ml per infusion over a1 sec period (Razel Scientific Instruments Inc., Stamford, Conn., USA),while responding on the inactive lever had no consequences. Rats wereconsidered to have acquired stable operant responding when they pressedthe active lever more than twice the number of times they pressed theinactive lever, received a minimum of six infusions or 90 pellets per1-h session, with less than 20% variation in the number of reinforcersearned per session. Rats took approximately 2 weeks to establish stablerates of operant responding. All test sessions were conducted for 1 hper day, 5 days per week.

Intracranial Self-Stimulation (ICSS) Reward Threshold Procedure (Markouand Koob, 1991; 1993)

Rats (n=9) were trained to respond according to a modification of thediscrete-trial current-threshold procedure of Kornetsky and Esposito(1979). Briefly, a trial was initiated by the delivery of anon-contingent electrical stimulus. This electrical reinforcer had atrain duration of 500 ms and consisted of 0.1 msec rectangular cathodalpulses that were delivered at a frequency of 50-100 Hz. The frequency ofthe stimulation was selected for individual animals so that baselinecurrent-intensity thresholds of each subject were within 50-200 □A, andthus allowed both threshold elevations and lowerings to be detected. Thefrequency was held constant throughout the experiment. A one-quarterturn of the wheel manipulandum within 7.5 sec of the delivery of thenon-contingent electrical stimulation resulted in the delivery of anelectrical stimulus identical in all parameters to the non-contingentstimulus that initiated the trial. After a variable inter-trial interval(7.5-12.5 sec, average of 10 sec), another trial was initiated with thedelivery of a non-contingent electrical stimulus. Failure to respond tothe non-contingent stimulus within 7.5 sec resulted in the onset of theinter-trial interval. Responding during the inter-trial interval delayedthe onset of the next trial by 12.5 sec. Current levels were varied inalternating descending and ascending series. A set of three trials waspresented for each current intensity. Current intensities were alteredin 5 μA steps. In each testing session, four alternatingdescending-ascending series were presented. The threshold for eachseries was defined as the midpoint between two consecutive currentintensities that yielded “positive scores” (animals responded for atleast two of the three trials) and two consecutive current intensitiesthat yielded “negative scores” (animals did not respond for two or moreof the three trials). The overall threshold of the session was definedas the mean of the thresholds for the four individual series. Eachtesting session was ˜30 min in duration. The time between the onset ofthe non-contingent stimulus and a positive response was recorded as theresponse latency. The response latency for each test session was definedas the mean response latency of all trials during which a positiveresponse occurred. After establishment of stable ICSS reward thresholds,rats were tested in the ICSS procedure once daily except for thetime-course of cocaine's lowering actions on ICSS thresholds when ratswere tested at time-points according to the experimental design.

Experiment 3.1: The Effects of MPEP Administration on CocaineSelf-Administration under a Fixed Ratio Schedule of Reinforcement

After training in the cocaine self-administration paradigm under a fixedratio (FR) schedule of reinforcement, as described above, two balancedgroups of rats were formed such that their rate of cocaineself-injections did not differ under baseline conditions. From day 10onwards (the ‘escalation’ period), access to cocaine self-administrationwas increased from 1 h to 6 h per daily session in one group of rats(Long Access or LgA rats; n=7). Previous studies have shown that thisschedule of access to cocaine self-administration results in aprogressive increase or ‘escalation’ in daily cocaine consumption (Ahmedand Koob, 1997; Ahmed et al. 2001). In the other group (Short Access orShA rats; n=7), access to cocaine self-administration was maintained at1 h per daily session. Previous studies have shown that this schedule ofaccess to cocaine self-administration maintains a stable level of dailycocaine consumption. After 22 days of 1 h (ShA) or 6 h (LgA) dailyaccess to cocaine self-administration, both groups of rats receivedtheir first MPEP injection. LgA and ShA rats were injected with MPEP (0,1, 3 or 9 mg/kg) according to a within-subjects Latin-square design, andthe daily cocaine self-administration session initiated 30 min later. Aminimum of 48 h were allowed between each injection in the Latin-squaredesign, during which LgA and ShA rats had their daily cocaineself-administration session, to ensure that rates of responding forcocaine returned to pre-injection baseline before the next MPEPadministration. After completion of the Latin-square, all rats receivedan injection of MPEP (6 mg/kg), and the daily cocaineself-administration session was initiated 30 min later.

Experiment 3. 2: The Effects of MPEP on Cocaine and NicotineSelf-Administration under a Progressive Ratio Schedule of Reinforcement

Under a fixed ratio schedule of reinforcement, as described above, ananimal responds a ‘fixed’ number of times on an active lever to obtain adrug infusion. Fixed ratio (FR) schedules of reinforcement provideimportant information on whether a drug is reinforcing. In contrast,under a progressive ratio (PR) schedule of reinforcement, each time ananimal responds on the active lever to receive a drug infusion, thenumber of times that the animal must subsequently respond to receive thenext infusion is progressively increased. By determining how hard ananimal is willing to work for a drug, while limiting total intake, thePR schedule allows better separation of motivation for drug consumptionfrom possible satiating effects of cumulative drug doses (Stafford etal. 1998). Such characteristics result in theoretically differentinterpretations of the factors controlling drug-seeking behavior on a PRcompared with a FR. For example, some researchers have recentlysuggested that FR schedules measure the pleasurable or hedonic effectsof a drug (McGregor and Roberts 1995; Mendrek et al. 1998), whereas PRschedules provide a better measure of the incentive or the ‘motivation’to obtain a drug (Markou et al., 1993). After acquisition of cocaine ornicotine self-administration under an FR, as described above, rats wereswitched to a PR schedule of reinforcement in which the followingsequence of level presses was required to receive each subsequentinfusion of nicotine or cocaine: 5, 10, 17, 24, 32, 42, 56, 73, 95, 124,161, 208, etc. Rats were injected with MPEP (0, 1, 3 or 9 mg/kg) 30 minprior to each PR session. All PR sessions lasted for 3 h. All subjectsreached breaking-points during the 3 hour session. Break-point isdefined as the highest ratio achieved before the session was terminated;the session was terminated if the subject failed to earn a drug infusionduring one hour.

Experiment 3.3: The Effects of MPEP on Cocaine-Induced Lowering of ICSSThresholds

MPEP was shown in Example 3.2 to decrease nicotine self-administration,under an FR, in rats and mice. The studies in this Example (resultsdescribed below) demonstrate that MPEP decreased cocaine and nicotineself-administration in rats (in the case of nicotine in mice also) undera FR, and decreased cocaine and nicotine self-administration in ratsunder a PR schedule of reinforcement. One possible mechanism by whichMPEP may have decreased cocaine self-administration behavior was bydecreasing the hedonic actions of cocaine. Cocaine-induced lowering ofICSS thresholds represents an accurate measure of cocaine's hedonic andeuphorigenic actions. Thus, to test the hypothesis that MPEP attenuatedthe hedonic actions of cocaine, we examined whether MPEP blockedcocaine-induced lowering of ICSS thresholds. The dose of cocaine (10mg/kg) used in these studies was chosen based on previous observationsthat this dose of cocaine induced maximal threshold lowering withoutaffecting performance in the ICSS procedure used in the present study(Kenny et al., 2002b; Markou & Koob 1992), and was equivalent to theamount of cocaine consumed by ShA rats during their daily 1 h access tococaine self-administration. Rats (n=9) were prepared with ICSSelectrodes as described above, and trained in the ICSS procedure untilstable thresholds were achieved (≦10% variation in thresholds over 5consecutive days). To determine if MPEP attenuated the magnitude ofcocaine's threshold-lowering effects, rats were injected with MPEP (0,3, 6 or 9 mg/kg) according to a within-subjects Latin-square design 30min prior to initiation of the ICSS session. All rats then received asaline injection 20 min later, 10 min prior to initiation of the ICSSsession. A period of 72 h were allowed to elapse between each injectionday in the Latin-square design, during which daily ICSS thresholdscontinued to be assessed to ensure that ICSS thresholds returned topre-injection baseline before the next drug administration. Aftercompletion of the Latin-square, all rats received an injection of MPEP(1 mg/kg), 30 min prior to initiation of the ICSS session, and a salineinjection 10 min prior to the initiation of the ICSS session. After thistreatment regimen, rats were once again injected with MPEP (0, 3, 6 or 9mg/kg) according to a within-subjects Latin-square design 30 min priorto initiation of the ICSS session. All rats then received a cocaine (10mg/kg) injection instead of saline 20 min later, 10 min prior toinitiation of the ICSS session. A period of 72 h were allowed betweeneach injection day in the Latin-square design, during which daily ICSSthresholds continued to be assessed to ensure that ICSS thresholdsreturned to pre-injection baseline before the next drug administration.After completion of the Latin-square, all rats received an injection ofMPEP (1 mg/kg), 30 min prior to initiation of the ICSS session, and acocaine (10 mg/kg) injection 10 min prior to the initiation of the ICSSsession.

To determine if MPEP attenuated the duration of cocaine'sthreshold-lowering effects, rats were first injected with either sterilewater or MPEP (3mg/kg), then 20 min later with either saline or cocaine(10 mg/kg) 10 min prior to initiation of the first ICSS session. Ratsreceived all injections according to a within-subjects Latin-squaredesign, such that each rat received the four possible drug combinations(water-saline; water-cocaine; MPEP-saline; MPEP-cocaine). lCSSthresholds were then assessed 10, 40, 70 and 100 min after the second(saline or cocaine) injection. A period of 72 h were allowed to elapsebetween each injection day in the Latin-square design, during whichdaily ICSS thresholds continued to be assessed to ensure that ICSSthresholds returned to pre-injection baseline levels before the nextdrug administration.

Experiment 3.4: Effects of the mGluR2/3 Antagonist on NicotineSelf-Administration under a Fixed Ratio Schedule.

Previously, the Group II metabotropic glutamate receptor antagonistLY341495 was shown to attenuate nicotine withdrawal, as measured bydecreased elevations in ICSS thresholds during spontaneous nicotinewithdrawal in rats (Kenny et al., 2003c). To examine whether mGlu2/3receptors also play a role in regulating the reinforcing actions ofnicotine, we examined whether LY341495 decreased nicotineself-administration under a FR schedule of reinforcement. After trainingin the nicotine self-administration paradigm, as described above, ratswere injected with LY341495 (0, 0.1, 0.5, 1, 3 or 5 mg/kg) according toa within-subjects Latin-square design, and the daily nicotineself-administration session was initiated 30 min later. A minimum of 48h were allowed between each injection in the Latin-square design, duringwhich rats had their daily nicotine self-administration session, toensure that rates of responding for nicotine returned to pre-injectionbaseline before the next LY341495 administration.

Experiment 3.5: The Effects of the Drug Combination LY341495 and MPEP onNicotine Self-Administration under a FR Schedule (Assessing Consumption)and a PR Schedule (Assessing Motivation for Drug Administration)

Previously, MPEP was shown to decrease nicotine self-administrationunder a FR schedule in rats and mice (Paterson et al., 2003). Thepresent studies demonstrate (results described below) that LY341495similarly decreased nicotine self-administration in rats under a FRschedule. Thus, we hypothesized that simultaneous inhibition of mGlu5and mGlu2/3 receptors, by MPEP and LY341495 respectively, may haveadditive inhibitory effects on nicotine self-administration in rats.Here we have shown (results described below) that LY341495 (0.5 mg/kg)decreased nicotine self-administration by approximately 35 percent.Thus: 1) we injected rats with this dose of LY341495 (0.5 mg/kg), incombination with a dose of MPEP (1 mg/kg) previously shown to have noeffects on nicotine (Paterson et al., 2003) or cocaine (present resultsfrom Experiment 1 reported here) self-administration, and assessednicotine self-administration behavior under a FR schedule ofreinforcement (30 minutes pretreatment); 2) we injected rats (30 minpretreatment) also with a dose of LY341495 (1 mg/kg) that was shown tohave no effects on nicotine self-administration when administered alonein combination with 1 mg/kg MPEP (this dose of MPEP was also shown tohave no effect on nicotine self-administration when administered alone)and assessed the effects of this drug combination treatment on nicotineself-administration; and 3) finally, we injected rats with the drugcombination treatment of 1 mg/kg LY341494 and 9 mg/kg MPEP (dose of MPEPthat decreases nicotine self-administration when administered on itsown; Paterson et al. 2003) and examined the effects on nicotineself-administration under a progressive ratio schedule of reinforcement(30 min pretreatment).

Statistical Analyses

During the escalation phase of the cocaine self-administrationexperiment, the number of cocaine responses in ShA and LgA rats duringthe 23 days prior to the first MPEP injection was analyzed by two-factorrepeated measures analyses of variance (ANOVA). During the MPEPtreatment phase, percent change from baseline number of cocaineresponses was calculated by expressing the number of cocaine responsesafter MPEP treatment as a percentage of the baseline number of cocaineresponses. For the ICSS experiments, mean raw thresholds and responselatencies (±SEM) are presented for each experiment in the resultssection. For all ICSS experiments, percentage change from baselinereward threshold was calculated by expressing the drug-influenced rawthreshold scores as a percentage of the baseline thresholds. Thebaseline thresholds were the mean of the thresholds obtained on thethree days before the first MPEP injection. For the first ICSSexperiment (the effects of MPEP on cocaine-induced threshold lowering),percentage of baseline scores were subjected to two-factorrepeated-measures ANOVA, with MPEP dose (1-9 mg/kg) and cocaine dose (0or 10 mg/kg) as the two within-subjects factors. For the second ICSSexperiment (the effects of MPEP on the duration of cocaine-inducedthreshold lowering), percentage of baseline scores were subjected tothree-factor repeated-measures ANOVA, with MPEP (0 or 3 mg/kg), cocaine(0 or 10 mg/kg) and time after second injection (10, 40, 70 and 100min), as the three within-subjects factors. For all ICSS experiments,response latency data were analyzed in the same manner as the thresholddata. The baseline number of nicotine responses was the mean number ofnicotine responses on the five days prior to the first LY341495injection. During the LY341495 treatment phase, percent change frombaseline number of nicotine responses was calculated by expressing thenumber of nicotine responses after LY341495 treatment as a percentage ofthe baseline number of nicotine responses. Progressive ratio data wereexpressed as highest ratio attained (i.e., break-point) or number ofinfusions earned (data expressed as raw values). After statisticallysignificant effects in the ANOVAs, post-hoc comparisons among means wereconducted with Fisher's LSD test. The level of significance was set at0.05.

Results

Experiment 3.1: The Effects of MPEP Administration on CocaineSelf-Administration under a Fixed Ratio Schedule of Reinforcement

As can be seen in FIG. 9A, the number of cocaine responses progressivelyincreased in LgA rats compared to ShA rats during the escalation phase.This effect was reflected in a statistically significant main effect ofDaily access (1 or 6 h) (F_((1,21))=10.96, p<0.01), a significant maineffect of Days of treatment (F_((12,252))=10.96, p<0.001), and asignificant Access×Days interaction (F_((12,252))=1.69, p<0.05). MPEP(1-9 mg/kg) decreased cocaine self-administration in ShA and LgA rats(F_((4,48))=9.34, p<0.001) (FIG. 9B). Post-hoc analysis demonstratedthat 3 (p<0.05), 6 (p<0.01) and 9 (p<0.001) mg/kg MPEP significantlydecreased cocaine responding in ShA rats (FIG. 9B). Post-hoc analysisdemonstrated that only the highest dose of MPEP (9 mg/kg) significantlydecreased cocaine responding in LgA rats (p<0.01) (FIG. 9B). However,there was no Dose×Access interaction (F_((12.252))=0.97, N.S.). Whencocaine responding for the ShA and LgA rats was collapsed, MPEPsignificantly decreased cocaine responding (F_((4,52))=9.36, p<0.001)(FIG. 9C), and post-hoc analysis demonstrated that 3 (p<0.01), 6(p<0.01) and 9 (p<0.001) mg/kg MPEP significantly decreased cocaineresponding in the collapsed group (FIG. 9C).

Experiment 3.2: The Effects of MPEP on Cocaine and NicotineSelf-Administration under a Progressive Ratio Schedule of Reinforcement.

A two-way ANOVA with MPEP dose and reinforcer as the two factorsrevealed a significant interaction effect of the two factors[F(6,48)=3.95, p<.01], a main effect of MPEP [F(3,48)=3.95; p<0.01] andno significant main effect of reinforcer [F(2,16)=1.41, p=0.27). As canbe seen in FIG. 10, MPEP (1-9 mg/kg) decreased responding for cocaine(significant effect of MPEP in a one-way follow-up ANOVA after asignificant interaction effect in the overall ANOVA: F(3,15)=12.76;p<0.001) and nicotine (significant effect of MPEP in a one-way follow-upANOVA after a significant interaction effect in the overall ANOVA:F(3,18)=1 1.28; p<0.001) under a progressive ratio schedule ofreinforcement, while having no statistically significant effect onresponding for food (no effect of MPEP on food responding in a one-wayfollow-up ANOVA after a significant interaction effect in the overallANOVA: F(3,15)=2.84; p=0.07. Because progressive ratio schedules ofreinforcement provide a measure of an animals ‘motivation’ to obtain thedrug, these data demonstrate that MPEP decreased the motivation of ratsto obtain cocaine or nicotine without affecting their motivation forfood, thus demonstrating the specificity of the effects.

Experiment 3.3: The Effects of MPEP on Cocaine-Induced Lowering of ICSSThresholds

As can be seen in FIG. 11A, cocaine (10 mg/kg) significantly loweredICSS thresholds (F_((1,8))=98.21, p<0.001). In contrast, MPEPsignificantly elevated ICSS thresholds (F_((4,32))=8.22, p<0.001), andpost-hoc analysis demonstrated that 6 (p<0.05) and 9 (p<0.01) mg/kg MPEPsignificantly elevated ICSS thresholds. There was no cocaine×MPEPinteraction (F_((4.32))=0.75, N.S.). Further analyses based on oura-priori hypotheses revealed that ICSS thresholds were significantlyelevated in cocaine-treated rats that previously received MPEP (9 mg/kg)compared to cocaine-treated rats that received vehicle injection(p<0.05) (FIG. 11A). However, ICSS thresholds in cocaine-treated ratsthat previously received MPEP (9 mg/kg) were still significantly loweredcompared to saline-treated rats pretreated with vehicle (p<0.001).Cocaine (10 mg/kg) significantly decreased ICSS response latencies(F_((1,8))=42.13, p<0.001) (FIG. 11B). In contrast, MPEP significantlyincreased response latencies (F_((4.32)=)2.8, p<0.05). Further analysisdemonstrated that only the highest dose of MPEP (9 mg/kg) significantlyincreased response latencies (p<0.05) (FIG. 11B). There was nococaine×MPEP interaction (F_((4,32))=2.16, N.S.).

As can be seen in FIG. 12A, cocaine (10 mg/kg) significantly loweredICSS thresholds (F_((1,8))=62.73, p<0.001). This cocaine-inducedlowering in ICSS thresholds was time-dependent; cocaine×time interaction(F_((3,24))=30.75, p<0.001). Post-hoc analysis revealed that ICSSthresholds were significantly lowered in cocaine-treated rats,pretreated with vehicle, at 10 (p<0.001) and 40 (p<0.001) min aftercocaine injection compared to saline-treated rats pretreated withvehicle. MPEP (3 mg/kg) had no effect on ICSS thresholds at anytime-point (F_((1,8))=0.002, N.S.), and there was no cocaine×MPEP×timeinteraction (F_((3,24))=1.02, N.S.). Post-hoc analysis revealed thatICSS thresholds were significantly lowered in cocaine-treated rats,pretreated with MPEP, at 10 (p<0.001) and 40 (p<0.001) min after cocaineinjection compared to vehicle-saline-treated rats. Cocaine significantlydecreased ICSS response latencies (F_((1,8))=12.65, p<0.01) (FIG. 12B).This cocaine-induced lowering in response latencies was alsotime-dependent; cocaine×time interaction (F_((3,24))=6.62, p<0.01).Post-hoc analysis revealed that response latencies were significantlydecreased in cocaine-treated rats, pretreated with vehicle, at 10(p<0.001) min after cocaine injection compared to saline-treated ratspretreated with vehicle. MPEP (3 mg/kg) had no effect on responselatencies at any time-point (F_((1,8))=2.67, N.S.), and there was nococaine×MPEP×time interaction (F_((3,24))=0.14, N.S.). Post-hoc analysisrevealed that ICSS thresholds were significantly lowered incocaine-treated rats, pretreated with MPEP, at 10 (p<0.001) min aftercocaine injection compared to vehicle-saline-treated rats.

Experiment 3.4: Effects of the mGluR2/3 Antagonist on NicotineSelf-Administration under a Fixed Ratio Schedule.

As seen in FIG. 13, LY341495 (0.1-5 mg/kg) decreased nicotineself-administration under a FR schedule (F_((5,35))=3.75, p<0.01).Post-hoc analysis demonstrated that 0.1 (p<0.01), 0.5 (p<0.05), 3(p<0.01) and 5 (p<0.01) mg/kg LY341495 significantly decreased nicotineresponding (FIG. 13).

Experiment 3.5: The Effects of the Drug Combination LY341495 and MPEP onNicotine Self-Administration under a FR Schedule and a PR Schedule.

As seen in FIG. 14, a combination of LY341495 (0.5 mg/kg) and a dose ofMPEP (1 mg/kg), shown in Example 2 above to have no effects on nicotine(See also, Paterson et al., 2003) or cocaine self-administration,significantly decreased responding for nicotine (F_((2,17))=8.6,p<0.01).

Post-hoc analysis demonstrated that LY341495 (0.5 mg/kg) alone (p<0.05),and the combination of LY341495 (0.5 mg/kg) and MPEP (1 mg/kg) (p<0.01)significantly decreased nicotine self-administration. The combination ofLY34,1495 (0.5 mg/kg) and MPEP (1 mg/kg) decreased nicotineself-administration by a greater magnitude that LY341495 (0.5 mg/kg)alone, although this effect just failed to reach statisticalsignificance (p=0.053). Similarly, a dose of LY341495 (1 mg/kg),combined with a behaviorally inactive dose of MPEP (1 mg/kg) alsodecreased nicotine self-administration (FIG. 15). Finally, additionaldata (FIG. 16) indicated that the combination of 9 mg/kg MPEP togetherwith 1 mg/kg LY341494 significantly decreased nicotineself-administration under a progressive ratio schedule of reinforcement.

Discussion

Recent observations from our laboratory have shown that repeatedextended (6 h) access cocaine self-administration resulted in aprogressive increase or ‘escalation’ in daily cocaine consumption in LgArats (Ahmed and Koob, 1998; Ahmed et al., 2002). In contrast, ShA ratswith restricted (1 h) access to cocaine self-administration maintained astable pattern of daily cocaine consumption. The present datademonstrate that the mGlu5 receptor antagonist MPEP decreased cocaineconsumption similarly in rats with ShA and LgA rats suggesting thatblockade of mGluR5 receptors may decrease drug use in bothdrug-dependent and non-drug-dependent individuals. Previously, MPEP wasshown to decrease cocaine self-administration in wild-type control mice(Chiamulera et al., 2001), and genetically modified mice in which themGlu5 deleted failed to acquire cocaine self-administration behavior,even though responding for food reinforcement was unaltered in thesemice (Chiamulera et al., 2001). Similarly, Example 2 above illustratesthat MPEP decreases nicotine self-administration in rats and mice. Thus,the data in this Example are consistent with an important role for mGlu5receptors in regulating cocaine and nicotine self-administrationbehavior in both drug-dependent and non-drug dependent individuals.However, MPEP decreased cocaine consumption by a similar magnitude inShA and LgA rats. Thus, it is unlikely that the escalation in cocaineintake observed in LgA rats was associated with alterations in mGlu5receptor regulation of cocaine self-administration behavior.

The hedonic actions of cocaine are thought to play an important role inestablishing and maintaining cocaine self-administration behavior(Stewart et al., 1984; Kenny et al., 2003a). Cocaine-induced lowering ofICSS thresholds is considered an accurate measure of cocaine's hedonicactions. Interestingly, doses of MPEP (1-9 mg/kg) that decreased cocaineself-administration did not attenuate the magnitude of the lowering inICSS thresholds observed after systemic cocaine administration.Similarly, MPEP (3 mg/kg) did not attenuate the duration of time thatcocaine lowered ICSS thresholds. Because cocaine-induced lowering ofICSS thresholds is an accurate measure of cocaine-induced facilitationof brain reward function, these observations suggest that MPEP decreasedcocaine consumption even though the hedonic impact of cocaine remainedintact. It is important to note that higher doses of MPEP (6-9 mg/kg)alone elevated ICSS thresholds, suggesting that mGlu5 receptorspositively regulate baseline brain reward function. However, a low doseof MPEP (3 mg/kg), that had no effect on ICSS thresholds alone,significantly decreased cocaine self-administration behavior. Thus, itis unlikely that MPEP decreased cocaine self-administration behavior bydecreasing baseline brain reward function and thereby inducing anaversive behavioral state. Overall, the data in this Example suggestthat MPEP decreased cocaine self-administration (i.e., consumption) by amechanism independent of cocaine's hedonic actions.

One possible explanation for the above observations is that MPEPdecreased the ‘motivation’ of rats to self-administer cocaine, but didnot alter cocaine-induced increases in brain reward function (i.e.,cocaine-induced euphoria). To test this possibility, we examined whetherMPEP would decrease responding for cocaine and nicotine under aprogressive ratio (PR) schedule of reinforcement. PR schedules provide ameasure of an animals ‘motivation’ to obtain a drug of abuse.Interestingly, MPEP significantly lowered the break point for cocaineand nicotine under a PR schedule of reinforcement. This observationsuggests that MPEP decreased the motivation of rats to obtain cocaineand nicotine, and thus leads to decreases in drug consumption.

Next, we examined whether blockade of mGlu2/3 receptors would decreasenicotine self-administration in rats similar to blockade of mGlu5receptors. Example 1 above, illustrates that blockade of mGlu2/3receptors by administration of the mGlu2/3 receptor antagonist LY341495,attenuated the depression-like aspects of nicotine withdrawal in rats.The present data demonstrated that LY341495, an antagonist at mGlu2/3receptors, decreased nicotine consumption in rats, suggesting thatblockade of mGluR2/3 receptors decreased the reinforcing effects ofnicotine. Most interestingly, co-administration of a dose of MPEP (1mg/kg) that has no effects on cocaine or nicotine self-administrationpotentiated the inhibitory effects of LY341495 (0.5 mg/kg or 1 mg/kg) onnicotine self-administration. Further, the combination of 9 mg/kg MPEPthat decreases either cocaine or nicotine self-administration whenadministered alone, when combined with 1 mg/kg LY341495, also decreasednicotine self-administration under a progressive ratio schedule ofreinforcement, and the effect was larger than with any one drug aloneclearly demonstrating a potentiation of these effects. Theseobservations suggest that simultaneous blockade of mGlu2/3 and mGlu5receptors may have better efficacy in the treatment of drug addictionthan blockade of either receptor subtype alone, and that suchsimultaneous blockade of mGlu2/3 and mGlu5 receptors decreases both drugconsumption and the motivation to engage in drug-taking behaviors.

Further, as illustrated in Example 1 above, blockade of mGluR2/3receptors reverses the affective depression-like aspects of nicotinewithdrawal. Thus, pharmacological treatments with dual antagonistactions at mGlu2 and/or mGluR3, and mGlu5 receptors: 1) decreaseconsumption of drugs of abuse, such as nicotine and cocaine to a largerextent than blockade of either one receptor alone and 2) reverse theaffective aspects of drug withdrawal, and possibly of drug dependence.These effects on both drug consumption and drug withdrawal maycontribute to increased abstinence rates among tobacco smokers, and drugusers and abusers. Finally, 3) pharmacological treatments with dualantagonist actions at mGlu2/3 and mGlu5 receptors may be effectivetreatments for non-drug-induced depressions also.

References Cited In Example 3

-   Chiamulera, C., Epping-Jordan, M. P., Zocchi, A., Marcon, C.,    Cottiny, C., Tacconi, S., Corsi, M., Orzi, F., Conquet, F., 2001,    Nat Neurosci, 4, 873-874.-   Esposito, R. U., Motola, A. H., Kornetsky, C., 1978, Pharmacol    Biochem Behav, 8, 437-439.-   Frank, R. A., Martz, S., Pommering, T., 1988, Pharmacol Biochem    Behav, 29, 755-758.-   Harris, G. C., Aston-Jones, G., 2003, Neuropsychopharmacology, 28,    73-76.-   Harrison, A. A., Liem, Y. T. and Markou, A. (2001),    Neuropsychopharmacology, 25,55-71.-   Kalivas, P. W., Duffy, P., 1998, J Neurochem, 70, 1497-1502.-   Kenny, P. J., Polis, I., Koob, G. F., Markou, A., 2003b, Eur J    Neurosci, 17, 191-195.-   Kenny P J, Gasparini F, Markou A. (2003c), J Pharmacol Exp Ther.    2003 Jun 12 [Epub ahead of print].-   Kokkinidis, L., McCarter, B. D., 1990, Pharmacol Biochem Behav, 36,    463-471.-   Laviolette, S. R., van der Kooy, D., 2003, Psychopharmacology    (Berl), 166, 306-313.-   Mansvelder, H. D., McGehee, D. S., 2000, Neuron, 27, 349-357.-   Markou, A. and Kenny, P. J. (2002), Neurotoxicity Research, 4(4),    297-313.-   Markou, A., Koob, G. F., 1992, Physiol Behav, 51, 111-119.-   Markou, A. and Koob, G. F. (1993) Intracranial self-stimulation    thresholds as a measure of reward. In: A. Sahgal (Ed.), Behavioural    Neuroscience: A Practical Approach, vol. 2, IRL Press, Oxford, pp.    93-115.-   Markou, A., Kosten, T. R. and Koob, G. F. (1998),    Neuropsychopharmacology, 18(3),135-174.-   McGeehan, A. J., Olive, M. F., 2003, Synapse, 47, 240-242.-   Popik, P., Wrobel, M., 2002, Neuropharmacology, 43,1210-1217.-   Stewart, J., de Wit, H., Eikelboom, R., 1984, Psychol Rev, 91,    251-268.-   Ungless, M. A., Whistler, J. L., Malenka, R. C., Bonci, A., 2001,    Nature, 411, 583-587.-   Wolf, M. E., 2003, Methods Mol Med, 79,13-31.

EXAMPLE 4 The Selective Serotonin Reuptake Inhibitor Paroxetine Combinedwith a Serotonin (5-HT)1a Receptor Antagonist Reversed Reward DeficitsObserved During Amphetamine Withdrawal in Rats

This example illustrates that the co-administration of the 5-HT1Areceptor antagonist p-MPPI and the selective serotonin reuptakeinhibitor paroxetine decreases the magnitude and reduces the duration ofamphetamine withdrawal-induced reward deficits.

Rationale: “Diminished interest or pleasure” in rewarding stimuli is anaffective symptom of amphetamine withdrawal, and a core symptom ofdepression. An operational measure of this symptom is elevation of brainstimulation reward thresholds during drug withdrawal. Data indicatedthat increasing serotonin neurotransmission by co-administration of theselective serotonin reuptake inhibitor (SSRI) fluoxetine and theserotonin-lA receptor antagonist p-MPPI reversed reward deficitsobserved during drug withdrawal (Harrison et al. 2001, incorporated inits entirety herein by reference). Objectives: We further tested thehypothesis that increased serotonergic neurotransmission would alleviatethis affective symptom of amphetamine withdrawal using paroxetine, amore selective SSRI than fluoxetine.

Methods: A discrete-trial current-threshold self-stimulation procedurewas used to assess brain reward function. The effects of paroxetine andp-MPPI alone and in combination were assessed in non-drug withdrawinganimals. We assessed also the effects of paroxetine and p-MPPI alone andin combination on reward deficits associated with amphetaminewithdrawal. Results: Paroxetine or p-MPPI alone had no effect onthresholds, while the co-administration of p-MPPI and paroxetineelevated thresholds in non-withdrawing rats. Amphetamine withdrawalresulted in threshold elevations. The co-administration of p-MPPI andparoxetine reduced the duration of amphetamine withdrawal-induced rewarddeficits.

Conclusions: Increased serotonergic neurotransmission decreased brainreward function in non-withdrawing rats, while the same treatmentreversed reward deficits associated with amphetamine withdrawal.Considering the greater selectivity of paroxetine compared to fluoxetinefor the serotonin transporter, these results strongly indicate that theaffective symptoms of amphetamine withdrawal, similarly tonon-drug-induced depressions, may be, in part, mediated through reducedserotonergic neurotransmission.

The withdrawal syndrome experienced after the cessation of amphetamineadministration is characterized by affective symptoms including“diminished interest or pleasure” in rewarding stimuli (AmericanPsychiatric Association 1994; Markou and Kenny 2002). The symptom of“diminished interest or pleasure” is also a core symptom of depressionand a negative symptom of schizophrenia (American PsychiatricAssociation 1994). Brain reward threshold elevation is an operationalmeasure of this symptom because it reflects reduced sensitivity torewarding electrical stimuli. In rats, withdrawal from a variety ofdrugs of abuse, belonging to diverse pharmacological classes such asnicotine (Epping-Jordan et al.1998; Harrison et al. 2001), amphetamine(Leith and Barrett 1976; Lin et al. 1999; Paterson et al. 2000; Harrisonet al. 2001), cocaine (Markou and Koob 1991), morphine (Schulteis et al.1994), ethanol (Schulteis et al. 1995) and phencyclidine (Spielewoy andMarkou 2003) elevated brain stimulation reward thresholds in rats.

Reduced serotonergic neurotransmission has been implicated in theetiology of non-drug induced depression. Evidence in favor of thishypothesis includes demonstrations of the efficacy of serotonergicantidepressant treatments, reduced cerebrospinal fluid levels ofserotonin metabolites, endocrine measures reflecting reducedserotonergic neurotransmission and the exacerbation of depressivesymptomatology seen after serotonin depletion in depressed patients (forreviews, Caldecott-Hazard et al. 1991; Markou et al. 1998). Recentadvances in the treatment of depression indicate that theco-administration of pindolol accelerates the delayed onset of theantidepressant action of selective serotonin reuptake inhibitors (SSRIs)(Rickels et al. 1989; Blier and de Montigny, 1999; Bordet et al. 1998;Zanardi et al. 1998; McAskill et al. 1998). It has been hypothesizedthat the acceleration of the antidepressant action of SSRIs may bethrough the 5-HT_(1A) receptor antagonist action of pindolol, althoughthis drug has widespread effects through antagonist actions at5-HT_(1A), 5-HT_(1E), and □-adrenergic receptors (Assie and Koek 1996;Bourin et al. 1998; Gobert and Millan 1999) and partial agonist actionsat □-adrenergic receptors (Clifford et al. 1998; Gobert and Millan 1999;Pauwels and Palmier 1994). In vivo microdialysis work demonstrated thatthe acute co-administration of a 5-HT_(1A) receptor antagonist togetherwith an SSRI rapidly elevated forebrain serotonin dialysate levelsbeyond levels seen after acute SSRI treatment alone (Auerbach and Hjorth1995; Hjorth 1993; Kreiss and Lucki 1995; Artigas et al 1996; Blier andde Montigny 1994; however, see Cremers et al. 2000).

We recently reported that the co-administration of4-(2′-methoxy-phenyl)-1-[2′-(n-(2″-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI), a5-HT_(1A) receptor antagonist (Kung et al. 1994), and fluoxetine, anSSRI (Wong et al. 1995), reversed reward deficits observed during eithernicotine or amphetamine withdrawal (i.e. increased reward) withoutaffecting the somatic signs of nicotine withdrawal (Harrison et al.2001). These data indicate that enhancement of serotonergicneurotransmission may selectively alleviate affective symptoms of drugwithdrawal. Further, the above results together with a related set ofstudies demonstrated that the effects of serotonergic manipulations onbrain reward mechanisms depend upon the hedonic state of the subjects(Harrison et al. 2001; Harrison and Markou 2001).

Paroxetine, a phenylpiperidine compound, acts as a selective serotoninreuptake inhibitor that is chemically and pharmacokinetically distinct,and more selective and potent in the inhibition of serotonin reuptakethan other SSRIs, such as fluoxetine, sertraline, clomipramine andfluvoxamine (Tulloch and Johnson 1992). SSRIs are hypothesized toalleviate the symptoms of depression through enhancement of serotonergictransmission (Tignol 1993; Bourin et al. 2001). However, SSRIs, such asfluoxetine, exert effects on the noradrenaline transporter also;noradrenaline is another transmitter system implicated in depression(Caldecott-Hazard et al. 1991; Markou et al. 1998). Thus, the purpose ofthe present study was to extend our previous findings with fluoxetine bydemonstrating that a more selective and chemically distinct SSRI,paroxetine, alleviates also the affective aspects of drug withdrawal andthus further demonstrates a serotonergic contribution to these effects.

In summary, based on evidence that: 1) pretreatment with a 5-HT_(1A)receptor antagonist potentiated the increase of forebrain serotoninlevels induced by paroxetine administration (Romero et al. 1996); 2)co-administration of pindolol with paroxetine accelerates the onset ofthe antidepressant effects of paroxetine in humans (Bordet et al. 1998;Zanardi et al. 1998), and 3) our previous findings that fluoxetinecombined with a 5-HT_(1A) receptor antagonist reversed amphetaminewithdrawal, it was hypothesized here that the co-administration of the5-HT_(1A) receptor antagonist p-MPPI with paroxetine would alleviateamphetamine withdrawal-induced reward deficits in rats. Thus, thepresent series of experiments assessed: 1) the effects of paroxetine onbrain reward thresholds under baseline conditions; 2) the effects ofp-MPPI and the co-administration of p-MPPI+paroxetine on thresholdsunder baseline conditions; and 3) the effects of these drug treatmentson reward deficits induced by amphetamine withdrawal.

Materials and Methods

Subjects

Male Wistar rats (Charles River, Hollister, Calif.) (300-320 g at thestart of the experiments) were housed in pairs in a temperature andhumidity controlled environment with a 12 hr light/dark cycle. Food andwater were available ad libitum. A different set of subjects was usedfor each experiment. All subjects were treated in accordance with theNational Institutes of Health “Guide for the Care and Use of LaboratoryAnimals,” and the animal facilities and the experimental protocols werein accordance with the Association for the Assessment and Accreditationof Laboratory Animal Care. Most of the behavioral testing was conductedduring the light phase of the subjects' light/dark cycle, unlessotherwise dictated by the experimental design due to time-courseassessment of behavioral parameters.

Apparatus

The experimental apparatus consisted of 16 Plexiglas chambers(30.5×30×17 cm) (Med Associates Inc., St. Albans, Vt.) encased insound-attenuating boxes (San Diego Instruments, San Diego, Calif.). Eachoperant chamber consisted of a stainless steel grid floor and a metalwheel manipulandum located on one wall, which required a 0.2 N force torotate it a quarter turn. Gold-contact swivel commutators and bipolarleads connected the animals in the stimulation circuit (Plastics One,Roanoke, Va.). Brain stimulation was administered by constant currentstimulators (Stimtek 1200, San Diego Instruments, San Diego, Calif.).

Surgical Procedure

The rats were prepared with 11 mm stainless steel bipolar electrodes(Plastics One; diameter=0.25 mm) in the posterior lateral hypothalamus(AP −0.5 mm from bregma; L±1.7 mm; DV −8.3 mm from dura, with theincisor bar set at 5 mm above the interaural line; Pellegrino et al.1979 under halothane anaesthesia (1-1.5% halothane/oxygen mixture).Subjects were allowed to recover for at least seven days before anybehavioral testing. Half of the electrodes were positioned on the righthemisphere and the other half on the left hemisphere to counterbalancepossible brain asymmetries.

Drugs

Paroxetine hydrochloride (generously provided by SmithKline Beecham,Worthing, West Sussex, U.K.) was dissolved in saline with a few drops ofpolyoxyethylenesorbitan monooleate (tween 80) (Sigma, St. Louis, Mo.)and then brought to a pH of approximately 6.5 using 0.05 M NaOH.Paroxetine was administered intraperitoneally in a volume of 4 ml/kg.4-(2′-Methoxy-phenyl)-1-[2′-(n-(2″-pyridinyl)-p-iodobenzamido]-ethyl-piperazinehydrochloride (p-MPPI) (Research Biochemicals Inc., Natick, Mass.) wasdissolved in sterile water and sonicated for 10-20 min in a heated waterbath, and then brought to a pH of approximately 5.2 with 0.1 M NaOH.p-MPPI was administered subcutaneously in a volume of 1 ml/kg.d-Amphetamine sulfate (obtained from the National Institute on DrugAbuse, Bethesda, Md.) was dissolved in saline and administeredintraperitoneally in a volume of 1 ml/kg.

Intracranial Self-Stimulation Behavioral Procedure

The ICSS discrete-trial current-threshold procedure is a modifiedversion (for details, see Markou and Koob 1992; Harrison and Markou2001) of a procedure initially developed by Kornetsky and coworkers(Kornetsky and Esposito, 1979). The subjects were initially trained toturn the wheel manipulandum on a fixed ratio 1 schedule ofreinforcement. The electrical reinforcer had a train duration of 500msec and consisted of 0.1 ms rectangular cathodal pulses delivered at afrequency of 100 Hz. The current intensity delivered was adjusted foreach animal and typically ranged from 100 to 200 μA. After successfulfamiliarization with this procedure (2 sessions of 100 reinforcers inless than 20 minutes), the rats were gradually trained on adiscrete-trial, current-threshold procedure.

At the start of each trial rats received a non-contingent electricalstimulus. During the following 7.5 sec, the limited hold, if thesubjects responded by turning the wheel manipulandum a quarter turn(positive response) they received a second, contingent stimulusidentical to the previous non-contingent stimulus. During a 2 sec periodimmediately following a positive response, further responses wererecorded as extra responses but had no consequence. If no responseoccurred during the 7.5 sec limited hold period a negative response wasrecorded. The inter-trial interval (ITI), which followed the limitedhold period, had an average duration of 10 sec (ranging from 7.5-12.5sec). Responses that occurred during the ITI were recorded as time-outresponses and resulted in a further 12.5 sec delay of the onset of thenext trial. Stimulation intensities were varied according to theclassical psychophysical method of limits. The subjects received fouralternating series of ascending and descending current intensitiesstarting with a descending series. Within each series the stimulusintensity was altered by 5 □A steps between each set of trials (threetrials per set). After training in the above procedure, rats were testeduntil stable baseline thresholds had been achieved L+10% over a 5-dayperiod). Drug testing commenced only after performance had stabilized,which typically occurred after two to three weeks of daily baselinetesting. Each test session typically lasted 30 minutes and provided twodependent variables for behavioral assessment:

Thresholds: The current-threshold for each descending series was definedas the stimulus intensity between a successful completion of a set oftrials (positive responses during two or more of the three trials) andthe stimulus intensity for the first set of trials, of two consecutivesets, during which the animal failed to respond positively on two ormore of the three trials. During the ascending series, the reversesituation defined the threshold. Thus, during each session, fourcurrent-thresholds were recorded and the mean of these values was takenas the current-threshold for each subject for each test session.

Response Latency: The latency between the onset of the non-contingentstimulus and a positive response was recorded as the response latency.The response latency for each test session was defined as the meanresponse latency of all trials during which a positive responseoccurred.

Expample 4.1

Effects of Paroxetine on Brain Stimulation Reward

The SSRI paroxetine (0, 1.25, 2.5, 5, 10 mg/kg; n=13) was administered120 min before the test sessions according to a within-subjects Latinsquare design with a minimum of seven days between drug injections toensure return to baseline levels prior to further drug testing.

Expample 4.2

Effects of p-MPPI and Combinations of p-MPPI+Paroxetine on BrainStimulation Reward

The effects of p-MPPI alone, and in combination with two doses ofparoxetine on brain stimulation reward were assessed using a factorialexperimental design. Dose of p-MPPI (0, 1, 3, 10 mg/kg) was thewithin-subject factor, and dose of paroxetine (0, 1.25 or 5 mg/kg; n=9:11 and 14 respectively) was the between-subject factor. p-MPPI wasadministered 135 min prior to test and paroxetine was administered 120min before test. There was a minimum of seven days between druginjections when paroxetine was administered, and a minimum of three daysbetween drug injections when p-MPPI was administered alone, to ensure areturn to baseline threshold levels prior to further drug testing.

Expample 4.3

Effects of Paroxetine, p-MPPI and a Combination of p-MPPI+Paroxetine onReward Deficits During Amphetamine Withdrawal

The amphetamine administration regimen used was a modification of thatused by Leith and Barrett (1976) and identical to that used by Lin etal. (1999) and Harrison et al. (2001). d-Amphetamine sulfate wasadministered intraperitoneally three times a day (6:00 A.M., 12:00 P.M.,6:00 P.M.) for four days in a rising dose regimen starting at 1 mg/kgand stabilizing at 5 mg/kg (i.e., 1, 2, 3, 4, 5, 5, 5, 5, 5, 5, 5, 5 mg/kg; total dose=50 mg/kg; n=46; 4 experimental groups, n=11-12/group).Another set of rats (n=43; 4 experimental groups, n=10-1 2/group) wasinjected at the same time points with saline. Body weight, intracranialself-stimulation reward thresholds and response latencies were measuredeach day during this chronic drug administration phase just prior to thefirst daily injection of either amphetamine or saline (i.e., 5:30 A.M.).Intracranial self-stimulation reward thresholds and response latencieswere then determined at 12, 36, 42, 60, 84, 108, 132 and 156 hr afterthe final amphetamine or saline injection. Based on the animals'performance during the 12 hr test after the final amphetamine or salineinjection, subjects were assigned to treatment groups so that originalwithdrawal effects on threshold elevations were equal across groups.Body weight was measured at 12, 36, 60, 84, 108, 132 and 156 hr afterthe final amphetamine or saline injection. Acute administration ofvehicle, p-MPPI (3 mg/kg), paroxetine (1.25 mg/kg), or p-MPPI (3mg/kg)+paroxetine (1.25 mg/kg) occurred prior to the 36 hr test session.This time point was selected based on the time course of thresholdelevations observed during amphetamine withdrawal previously (Lin et al.1999; Paterson et al. 2000; Harrison et al. 2001). Doses of paroxetineand p-MPPI were selected based on the results of experiments 1 and 2.p-MPPI was administered 135 min prior to test, and paroxetine wasadministered 120 min prior to test.

Data Analyses

In Example 4.1 and 4.2 reward thresholds and response latencies wereexpressed as a percentage of the mean baseline values assessed duringthe three days prior to each drug treatment. Data from Example 4.1 wereanalyzed using a one-way repeated measures Analysis of Variance (ANOVA)and a linear trend analysis. Data from Example 4.2 were analyzed using atwo-way mixed factors ANOVA, with doses of p-MPPI as the within-subjectfactor and doses of paroxetine as the between-subject factor. Due to thestrong a priori hypothesis that p-MPPI+paroxetine drug combinationswould elevate reward thresholds (Harrison and Markou 2001), furtheranalysis of each dose-response curve was conducted using linear trendanalysis (Hinkle et al. 1998). In Example 4.3 all reward threshold andresponse latency data were expressed as a percentage of the meanbaseline values during the five days immediately prior to the firstamphetamine or saline injection. Body weight data were expressed as apercentage of the weight immediately before the first amphetamine orsaline injection. Data collected during chronic drug treatment wereanalyzed using two-way mixed factors ANOVA. The within-subject factorwas days of treatment and the between-subject factor was chronic drugtreatment (saline or amphetamine). Data collected during amphetamine (orsaline) withdrawal were analyzed using three-way mixed factors ANOVA.The within-subject factor was time after amphetamine or salinetreatment, and the two between-subject factors were chronic drugtreatment (amphetamine or saline) and acute drug treatment administeredduring withdrawal. Statistically significant interactions were followedby post-hoc Newman-Keuls tests. The level of significance was set atp<0.05. All statistical analyses were conducted using the BMDPstatistical software package (BMDP Statistical Software Inc., Calif.).

Results

Baseline Thresholds and Response Latencies

Baseline data from Example 4.1 and Example 4.2 were analyzed usingone-way ANOVAs to assess potential drifts of baseline performance. Nosignificant differences in thresholds or response latencies were foundin any of the experiments. Example 4.1: range of mean thresholds:110.62-123.52 μA; range of mean response latencies: 3.17-3.34 sec.Example 4.2: p-MPPI+ vehicle: range of mean thresholds: 110.29-113.73μA; range of mean response latencies: 3.15-3.26 sec; p-MPPI+paroxetine(1.25 mg/kg): range of mean thresholds: 130.22-132.28 μA; range of meanresponse latencies: 3.25-3.27 sec; p-MPPI+paroxetine (5 mg/kg): range ofmean thresholds: 129.19-134.39 pLA; range of mean response latencies:3.29-3.37 sec. Example 4.3: There were no statistically significantdifferences between the mean baseline thresholds, response latencies orbody weights of subjects assigned to the saline “withdrawal” group(n=43) [mean threshold±SEM: 139.18±5.45 μA; mean response latency±SEM:3.46+0.06 sec; mean body weight±SEM: 508.83±7.40 g], and subjectsassigned to the amphetamine withdrawal group (n=46) [mean threshold±SEM:133.17±4.97 μA; mean response latency±SEM: 3.41±0.06 sec; mean bodyweight±SEM: 513.99±7.13 g]. Based on the animals' performance during the12 hr test after the final amphetamine or saline injection, subjectswere assigned to treatment groups so that original withdrawal effects onthreshold elevations were equal across groups.

Example 4.1

Effects of Paroxetine on Brain Stimulation Reward

Paroxetine had no statistically significant effect on thresholds[F(4,48)=1.77, n.s.] or response latencies [F(4,48)=2.16, n.s.] at thedoses administered in the present study (FIGS. 17A and 17B). A lineartrend analysis was not significant (p>0.05).

Example 4.2

Effects of p-MPPI and Combinations of p-MPPI+Paroxetine on BrainStimulation Reward

Analysis of the threshold data revealed a main effect of paroxetine[F(2,31)=22.22, p<0.01] and a main effect of p-MPPI [F(3,93)=5.65,p<0.05] but no significant interaction between the two drugs[F(6,93)=0.33, n.s.]. The a priori hypothesis that administration ofp-MPPI+paroxetine would elevate thresholds, based on previous findingswith p-MPPI+fluoxetine administration (Harrison and Markou 2001),permitted further analysis of these data. Linear trend analysis of eachdose-response curve revealed that p-MPPI administered alone had noeffect on reward thresholds [F(1,5)=3.748, n.s.], whereas p-MPPIadministered in combination with paroxetine (1.25 mg/kg or 5 mg/kg)elevated thresholds in a p-MPPI dose-related manner [F(1,7)=7.75, p<0.05and F(1,10)=7.95, p<0.05, respectively for the two doses of paroxetine](FIG. 18A). Analysis of the response latency data demonstrated thatparoxetine increased response latencies [F(2,31)=9.41, p<0.01], whereasp-MPPI had no effect on response latencies [F(3,93)=0.17, n.s.] and didnot interact with the effect of paroxetine on response latencies[F(6,93)=1.28, n.s] (FIG. 18B).

Example 4.3

Effects of Paroxetine, p-MPPI and a Combination of p-MPPI+Paroxetine onReward Deficits During Amphetamine Withdrawal

Chronic Amphetamine: Chronic amphetamine administration (four daytreatment) significantly elevated thresholds in compared to thresholdsof saline-exposed animals during the tests that were conductedimmediately before the first daily injection (12 hr after the previousamphetamine or saline injection) [F(1,87)=117.95, p<0.01]. Analysis of asignificant days of treatment×chronic drug treatment interaction[F(3,261 )=50.81, p<0.01 ] demonstrated that the thresholds ofamphetamine-exposed animals were elevated in a treatmentduration-related manner with significant elevations observed betweendrug days 1 (prior to the first injection; baseline) and 2, and drugdays 2 and 3, but not between drug days 3 and 4. The thresholds of theamphetamine-exposed animals were significantly higher than those of thesaline-exposed animals on drug days 2, 3 and 4. The thresholds ofsaline-exposed rats remained stable during this phase of the experiment(FIG. 19A).

Analysis of the response latency data revealed a significant days oftreatment×drug treatment interaction [F(3,261)=2.82, p<0.05]. Theresponse latencies of amphetamine-exposed animals were significantlyslower on drug day 2 when compared to drug day 1 (day prior totreatment; baseline), whereas the response latencies of saline-exposedanimals remained stable during treatment. Nevertheless, the responselatencies of the amphetamine-exposed rats did not significantly differfrom those of the saline-exposed rats at any stage during chronic drugtreatment (FIG. 19B).

Chronic amphetamine administration significantly reduced the body weightof animals in comparison to weights of saline-exposed controls[F(1,87)=282.5, p<0.01]. Analysis of a significant days oftreatment×drug treatment interaction [F(3,261)=209.77, p<0.01]demonstrated that the body weight of amphetamine-exposed rats wasreduced in a treatment duration-dependent manner with significantreductions of body weight observed on each day of chronic amphetaminetreatment compared to the previous day. By contrast the body weight ofsaline-exposed animals significantly increased between drug days 2 and3. The body weights of amphetamine-exposed animals were significantlylower than those of the saline-exposed animals on drug days 2, 3 and 4(FIG. 19C).

Amphetamine withdrawal: Amphetamine-exposed animals exhibited elevatedthresholds relative to saline-exposed rats after the final amphetamineinjection [F(1,81)=84.88, p<0.01]. Analysis of the significanttime×chronic treatment×acute treatment interaction [F(21,567)=1.63,p<0.05] revealed the following. Amphetamine-exposed rats treated withvehicle prior to the 36 hr time point exhibited elevated thresholds atwithdrawal hours 12, 36, 42, 60 and 84, that returned to baseline levels108 hr after the final amphetamine injection compared to saline-exposedrats treated with vehicle (FIG. 20A and 20D). Neither paroxetine (FIG.4A & 4D) nor p-MPPI (FIG. 20B and 20E) had an effect on thresholds ofsaline-exposed animals. However, both paroxetine (FIG. 20D) and p-MPPI(FIG. 20E) shortened the duration of the reward deficits as indicated byreturn to baseline reward threshold levels (defined as no statisticallysignificant differences from the saline-exposed vehicle-treated rats) 24hour before the amphetamine-withdrawing non-drug-treated rats. Theco-administration of p-MPPI+paroxetine resulted in significantlyelevated thresholds at the 36 hour time-point immediately after the drugadministration and significantly lower thresholds at the 60 hourtime-point compared to amphetamine-exposed vehicle-treated rats (FIG.20F). This combination treatment shortened the duration of the rewarddeficits as indicated by return to baseline reward threshold levels(defined as no statistically significant differences from thesaline-exposed vehicle-treated rats) 48 hour before theamphetamine-withdrawing non-drug-treated rats (FIG. 20F). The samecombination treatment had no significant effect in saline-exposed ratswhen compared to saline-exposed vehicle-treated rats (FIG. 20C).However, there was a trend for threshold elevations induced by thistreatment at the 36 hour time-point immediately after the drugadministration compared to later performance of the same group (i.e.,time points 84, 108, 132, and 156 hr; FIG. 20C).

Analysis of response latencies during withdrawal revealed a significanttime×acute treatment interaction [F(21,567)=4.00, p<0.01]. Acute vehicleor p-MPPI treatment had no effect on response latencies. However, theresponse latencies of paroxetine, and p-MPPI +paroxetine-treated animalswere significantly slower than those of either vehicle or p-MPPI treatedrats 36 hr after the final amphetamine or saline injection (FIG. 21).

Amphetamine-exposed animals had lower percentage body weights relativeto saline-exposed rats after the final amphetamine injection[F(1,81)=427.12, p<0.01; FIG. 22]. Analysis of a significanttime×chronic treatment×acute treatment interaction [F(18,486)=1.69,p<0.05] revealed the following. Both saline-exposed andamphetamine-exposed rats treated with vehicle significantly increased inbody weight after cessation of chronic drug administration asdemonstrated by significant increases of body weight between time points12 hr and 132 hr after the final injection (FIG. 22A).Amphetamine-exposed rats treated with p-MPPI alone appeared to gainweight similarly to vehicle-treated rats (FIG. 22B). Amphetamine-exposedrats treated with paroxetine alone did not gain weight during withdrawal(FIG. 22C). Most interestingly, amphetamine-exposed rats treated withthe drug combination started gaining weight faster than their respectivecontrols (i.e., saline-exposed rats treated acutely withp-MPPI+paroxetine (FIG. 22D).

Discussion

Acute administration of paroxetine, an SSRI, had no significant effecton reward thresholds or response latencies at the doses tested (FIGS.17A and 17B). However, slight non-significant elevations of rewardthresholds can be seen following the administration of doses above 1.25mg/kg, whereas paroxetine had less consistent effects on responselatencies. Administration of SSRIs have produced either small (10%) orno effects on reward thresholds and response latencies (Harrison andMarkou 2001; Harrison et al. 2001; Lin et al. 1999; Lee and Kornetsky1998; Katz and Carroll 1977). This small elevation of reward thresholdsinduced by SSRI administration may be related to non-specific effects ofthese drugs on motor performance, or decreased reward resulting from thepharmacological enhancement of serotonergic neurotransmission.

The administration of p-MPPI, a 5-HT_(1A) receptor antagonist, had noeffect on brain reward thresholds or response latencies whenadministered alone. By contrast, the co-administration ofp-MPPI+paroxetine (1.25 mg/kg or 5 mg/kg), doses of paroxetine that hadno effect on thresholds when administered alone, significantly elevatedthresholds in a p-MPPI dose-related manner. These drug combinations hadno effect on response latencies indicating a p-MPPI-induced potentiationof the effects of paroxetine on brain stimulation reward rather thanalterations in the animals' ability to perform the operant response.These data are consistent with previous reports that 5-HT_(1A) receptorantagonism, by p-MPPI, had no effects on brain stimulation reward whenadministered alone, yet potentiated the reward-reducing effects ofanother SSRI, fluoxetine (Harrison et al. 2001; Harrison and Markou2001). The reward reducing effects of the co-administration of a 5-HTIAreceptor antagonist and an SSRI may be due to p-MPPI-inducedaugmentation of the paroxetine-evoked increase in extracellularserotonin (Bel and Artigas 1993; Blier and de Montigny 1994; Hjorth1993). On the basis of these results indicating that p-MPPI augments theeffects of paroxetine (1.25 mg/kg) on brain stimulation reward, thisdose of paroxetine was administered either alone or in combination withp-MPPI (3 mg/kg) in an attempt to alleviate amphetaminewithdrawal-induced reward deficits.

During chronic drug treatment, brain stimulation reward thresholds weremeasured before the first daily injection of either amphetamine orsaline (on drug days 2, 3 and 4; this testing occurred 12 hr after thelast injection on the previous day). Reward thresholds ofamphetamine-exposed animals were significantly elevated on drug days 2,3 and 4 compared to those of the saline-exposed rats. The rewardthresholds of the amphetamine-exposed animals increased in a treatmentduration-dependent manner indicating that measurement of rewardthresholds, at this time each day, permitted the assessment of theprogressive formation of amphetamine withdrawal-induced reward deficits.However, the progressive nature of the formation of these rewarddeficits was limited. This is indicated by the lack of increases in themagnitude of the reward deficits after 2 days of amphetamineadministration (drug day 3) compared to after three days of treatment(drug day 4) (FIG. 19A). This result is consistent with previous reportsof limitations of the magnitude of reward threshold elevations after avariety of amphetamine administration regimens (Lin et al. 1999;Paterson et al. 2000). Chronic amphetamine administration had no effecton response latencies compared to those of saline control animalsindicating that the elevations of reward thresholds were due to aspecific effect on reward and not related to alterations in the animal'sability to perform the operant response.

Chronic amphetamine administration resulted in a treatmentduration-dependent reduction of body weight. A progressive reduction ofbody weight was observed after every day of amphetamine treatment. Bycontrast, animals chronically treated with saline increased in bodyweight during this phase of the experiment (see FIG. 18C). These dataare consistent with reports of the anorectic effects of amphetamine(Caul et al. 1988). In contrast to the reduction of body weight observedduring amphetamine administration, chronic nicotine administrationsuppressed body weight gain, resulting in no change of body weightduring the nicotine administration period, compared to saline-exposedcontrols that increased in weight over time (Harrison et al. 2001).Although the present study did not address the cause of weight loss, ithas been reported that amphetamine administration may reduce body weightby altering food consumption, metabolic rate and fat metabolism (Caul etal. 1988; Jones et al. 1992). Alternatively, the reduction of bodyweight observed during chronic amphetamine administration in the presentstudy may be the result of a disruption of behavior due to drug-inducedstereotypy which has been reported to be augmented by multiple dailyamphetamine injections (Segal et al. 1980).

During amphetamine withdrawal animals increased in body weight at asimilar rate to saline-exposed control rats (FIG. 22A). These data areconsistent with previous reports of similar increases in body weight ofamphetamine-withdrawing and saline control rats (Mucha et al. 1990).Acute paroxetine treatment prevented weight gain during the 7 days ofamphetamine withdrawal. It has been reported that in rats a single highdose of paroxetine (120-300 mg/kg) administered orally produces areduction in body weight and food consumption (Ryan et al. 2001). Thus,the failure of the paroxetine-treated animals to gain weight duringamphetamine withdrawal may be the result of this anorectic effect ofparoxetine treatment. By contrast, the co-administration ofparoxetine+p-MPPI resulted in earlier weight gain during amphetaminewithdrawal compared to non-withdrawing animals and to vehicle-treatedamphetamine-withdrawing animals. These data suggest that theco-administration of a 5-HTlA receptor antagonist with paroxetinefacilitated the reversal of amphetamine withdrawal-induced weight loss,whereas the administration of paroxetine alone prevented (during 7 days)or delayed weight gain during amphetamine withdrawal.

In replication of previous findings, amphetamine withdrawal resulted ina reward deficit, reflected in elevated brain reward thresholds relativeto saline-exposed rats and to pre-drug baseline thresholds (Leith andBarrett 1976; Kokkinidis and Zacharko 1980; Harrison et al. 2001; Lin etal. 1999; Paterson et al. 2000). Acute p-MPPI or paroxetineadministration had no consistent effect on withdrawal-induced rewarddeficits, but paroxetine significantly increased response latencies ofboth amphetamine- and saline-exposed rats. Acute administration ofp-MPPI+paroxetine reduced the duration of the withdrawal-induced rewarddeficits by 48 hr indicating a rapid return of sensitivity to therewarding electrical stimulation in the amphetamine-withdrawing animals.In contrast to the rapid alleviation of amphetamine-withdrawal inducedreward deficits, the same combination drug treatment appears to haveaugmented amphetamine-withdrawal reward deficits during the 36 hr testsession (2 hours after administration of the combination drug treatment)as indicated by elevated reward thresholds compared toamphetamine-withdrawing vehicle-treated rats. Similarly, thiscombination treatment resulted in a small and transient elevation ofreward thresholds (i.e. a decrease in reward) in saline-exposed ratsduring the 36 hr test.

Acute paroxetine and p-MPPI+paroxetine treatment reduced the speed ofresponding during the test session 2 hr after drug treatment in bothamphetamine- and saline-withdrawing rats. In the case ofparoxetine-treated animals this decrease in the speed of responding doesnot appear to have affected reward thresholds of eitheramphetamine-withdrawing or saline control animals. However, the apparentaugmentation of amphetamine withdrawal-induced deficits and elevationsof reward thresholds (i.e. reduced reward) of the saline-exposed animalstreated with the drug combination may be related to a non-specificeffect of this drug treatment on performance of the behavioral task.Although previous findings have indicated that reward thresholds are avalid and reliable measure of reward that are minimally affected byperformance manipulations (Markou and Koob, 1992), when elevations inreward thresholds are accompanied by a reduction of the speed ofresponding (i.e., increased response latency) it is possible that theseresults are related to disruption of performance rather than alterationsof brain reward function. The present data are strikingly similar to theeffects of fluoxetine, another SSRI, in the same experimental procedure(Harrison et al. 2001; Harrison and Markou 2001). In both cases acuteadministration of the SSRI reduced the duration of amphetaminewithdrawal-induced elevations of reward thresholds by 24 hr, an effectthat was dramatically augmented by the co-administration of a 5-HT_(1A)receptor antagonist with the SSRI. Further, in contrast to the rapidalleviation of reward deficits observed during amphetamine withdrawal,the combination of either SSRI and a 5-HT_(1A) receptor antagonistreduced the sensitivity of control animals to the rewarding electricalstimulation. One difference between these studies is the doses of theSSRI used to alleviate the reward deficits. When administered alone, 5mg/kg fluoxetine reduced the duration of the reward deficits, but 2.5mg/kg fluoxetine did not. The co-administration of p-MPPI with eitherdose of fluoxetine reversed the reward deficits observed duringamphetamine withdrawal. In the present study 1.25 mg/kg paroxetineproduced similar results to 5 mg/kg fluoxetine. However, in clinicalpractice, similar doses of these drugs are prescribed for the treatmentof depression suggesting similar antidepressant potency of these twodrugs in humans (Tignol 1993; Nemeroff 1993; Dunner and Dunbar 1992;Chouinard et al. 1999; Wagstaff et al. 2002). The present data indicatethat paroxetine may be more potent than fluoxetine in reversing thereward deficits associated with amphetamine withdrawal. These datatogether with previous reports that paroxetine is a more selective andpotent serotonin reuptake inhibitor than fluoxetine (Tulloch andJohnson, 1992) suggest that reduced serotonergic transmission plays animportant role in the mediation of psychostimulant withdrawal-inducedreward deficits. This hypothesis is consistent with in vivomicrodialysis data indicating reduced serotonergic transmission duringwithdrawal from drugs of abuse (Parsons et al. 1995; Weiss et al. 1996).Although these data do not preclude a role of noradrenergic transmissionin reward deficits associated with psychostimulant withdrawal, thepresent data offer strong support to the serotonergic hypothesis.

The rapid restoration of the sensitivity to the electrical stimulationobserved following the co-administration of either paroxetine orfluoxetine and a 5-HT_(1A) receptor antagonist may be attributable toincreased serotonergic transmisson in forebrain structures such as thefrontal cortex, hippocampus and the striatum (Bel and Artigas 1993:Dreshfield et al. 1996; 1997; Invernizzi et al. 1994; Gobert and Millan1999). These data are consistent with the hypothesis that the rapidonset of action of the clinical antidepressant action of SSRIs whencombined with pindolol (Bordet et al 1998; Tome et al 1997a; 1997b;Zanardi et al 1998; however, see Berman et al 1999) is partlyattributable to pindolol's 5-HT_(1A) receptor antagonist properties.However, other receptors such as 5-HT_(1B) and □-adrenergic receptorsmay also contribute to pindolol's augmentation of SSRI antidepressanteffects (see Introduction).

In contrast to the short lasting elevations of reward thresholds incontrol rats after the combination drug treatments, which is typical ofacute drug treatments, these acute treatments permanently reversedwithdrawal-induced reward deficits. This result is perhaps moresurprising after acute paroxetine treatment than after fluoxetinetreatment. Unlike fluoxetine which has a long half-life due to itsactive metabolite norfluoxetine, paroxetine has a half- life ofapproximately 24 hours (Lemberger et al. 1985; Prakash and Foster 1999).Therefore, the effectiveness of these acute treatments in reversingwithdrawal-induced reward deficits may be related to the limitedduration of the withdrawal-induced reward deficits. Thus, over time thedeclining enhancement of serotonergic neurotransmission, induced by theacute drug treatments, may coincide with the gradual return of rewardthresholds to baseline levels as withdrawal symptoms diminish. Further,the reward deficits in these experiments were transiently induced bydrug manipulation (amphetamine withdrawal) in healthy subjects. Thus,acute treatments may be more effective in reversing such reward deficitsin healthy subjects in comparison to similar deficits related to achronic imbalance of the brain reward system.

The elevation of reward thresholds in saline-exposed control rats afterthe co-administration of an SSRI and a 5-HT_(1A) receptor antagonistappear to be incompatible with the hypothesis that enhancement ofserotonergic transmission by antidepressant treatment elevates mood andalleviates reward deficits in depressed individuals (present data;Harrison et al. 2001; Harrison and Markou 2001). This apparentdiscrepancy is also observed in the present data between the effects ofsuch treatment on brain reward function of amphetamine-withdrawing rats(increased reward) and control rats (decreased reward). These datasuggest that the effects of enhanced serotonergic transmission on brainreward function are dependent upon the “hedonic” state of the subjectsat the time of treatment (Ahmed and Kood 1998; Koob and LeMoal 1997;Harrison et al. 2001; Harrison and Markou 2001).

In summary, the present data indicated that the co-administration of a5-HT_(1A) receptor antagonist with paroxetine potentiated the brainstimulation reward-reducing effects of the SSRI in “normal” subjects. Incontrast to the reward-reducing effects induced by the co-administrationof paroxetine and a 5-HT_(1A) receptor antagonist in “normal” subjects,the same drug combination alleviated brain stimulation reward deficitsobserved during amphetamine withdrawal (increased reward in anhedonicsubjects). These data with paroxetine extend the previous data withfluoxetine by indicating that a second chemically andpharmacokinetically distinct SSRI with higher selectivity for theserotonin tranporter reverses reward deficits associated withamphetamine withdrawal. Thus, these data enhance the predictive validityof amphetamine withdrawal-induced elevations of reward thresholds as amodel of depression. Furthermore, these SSRI data (present data;Harrison et al. 2001) support the hypothesis that reduced serotonergictransmission may be one of the neurobiological abnormalities underlyingthe symptom of “diminished interest or pleasure” seen during amphetaminewithdrawal (American Psychiatric Association 1994; Markou et al. 1998;Harrison et al. 2001). Further, considering the substantial evidencesuggesting that reduced serotonergic transmission is associated withnon-drug-induced depression, the present data may indicate similaritiesin the neurobiological substrates of both drug- and non-drug-induceddepression (Markou et al. 1998; Markou and Kenny 2002; Barr et al.2002). Specifically, reduced serotonergic transmission may underlie, inpart, the affective symptom of “diminished interest or pleasure” in bothof these types of depression.

References cited in Example 4

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EXAMPLE 5 Bupropion Enhances Brain Reward Function and Reverses theAffective and Somatic Aspects of Nicotine Withdrawal in the Rat

This example illustrates that the antidepressant bupropion enhancesbrain reward function and reverses the affective and somatic aspects ofnicotine withdrawal.

Rationale: Bupropion is an atypical antidepressant and the onlynon-nicotine-based therapy approved for smoking cessation. Its use hasraised much debate as to how a non-nicotine-based agent can aid insmoking cessation.

Objectives: We assessed the effects of bupropion on brain rewardfunction under baseline conditions and subsequent to withdrawal fromchronic nicotine administration in rats. Methods: A discrete-trialintracranial self-stimulation paradigm procedure was used that providesone with current intensity thresholds, a measure of reward in rats underbaseline conditions and subsequent to withdrawal from chronic nicotine(3.16 mg/kg per day for 7 days via osmotic minipump). Somatic signs wererecorded based on a checklist of nicotine abstinence signs in animalswithdrawn from nicotine. Results: Bupropion (10-60 mg/kg)dose-dependently lowered reward thresholds in non-withdrawing subjectsindicating an increase in reward. Interestingly, a sub-effective dose ofbupropion (5 mg/kg) blocked completely the threshold lowering effects ofacute nicotine (0.25 mg/kg). Animals withdrawn from chronic nicotineexhibited increases in somatic signs of withdrawal and elevated brainreward thresholds, which is indicative of “diminished interest orpleasure” (i.e. anhedonia) in the rewarding stimuli. Bupropion (10-40mg/kg) reversed both the reward deficit and the somatic signs, with thehighest dose (40 mg/kg) inducing a protracted reversal of the thresholdelevation.

Conclusions: Bupropion acts on multiple levels to alter brain rewardcircuits influenced by nicotine, in addition to reducing the expressionof somatic signs of withdrawal. First, bupropion, unlike otherantidepressants, increases brain reward function under baselineconditions in non-withdrawing subjects. Second, at low doses bupropionblocks the rewarding effects of nicotine. Third, bupropion reverses thenegative affective aspects of nicotine withdrawal. Such actions arelikely to act in concert to mediate the unique anti-smoking propertiesof bupropion.

Introduction

Tobacco smoking is a major public health problem worldwide. Estimates oftobacco related deaths account for 20% of all deaths in developedcountries (Peto et al. 1992). Accumulating evidence indicates thatnicotine is one of the active ingredients in tobacco smoke that leads toand maintains tobacco addiction (Stolerman and Jarvis 1995). Despite thefact that as many as 40% of smokers attempt to quit each year, onlyabout 6% of these succeed in maintaining abstinence (Jorenby et al.1999). Therefore, there is great impetus to understand theneurobiological mechanisms underlying both nicotine addiction andtherapies that are currently used in smoking cessation programs.

Two types of pharmacological therapies have been approved for smokingcessation by the Food and Drug Administration of the United States(Hughes et al. 1999; Glover and Glover 2001). The first are the nicotinereplacement therapies that allow the smoker to substitute the nicotinefrom cigarettes with other safer nicotine formulations such as chewinggum, transdermal patches, or inhalers (Hughes et al. 1999). The secondtherapy that is non-nicotine based is the atypical antidepressantbupropion (Hurt et al. 1997; Jorenby et al 1999; Hays et al. 2001;Tashkin et al. 2001; Ahluwalia et al. 2002; George et al. 2002). Whilstthe rationale behind the use of nicotine replacement therapies isintuitive, it remains unclear why bupropion is effective in thisindication. Bupropion's utility was first realized serendipitously bythe clinical observations that depressed patients on the drug decreasedtheir tobacco smoking (Balfour 2001). A possible rationale for theeffectiveness of bupropion is provided from the multitude of clinicalstudies showing a strong link between negative affect and the propensityto smoke and difficulty in quitting (Breslau et al. 1992;1998; Lipkus etal. 1994; Laje et al. 2001). Indeed, among smokers, symptoms of nicotinedependence are correlated with the magnitude of affective depressivesymptomatology (Anda et al. 1990; Glassman et al. 1990). However, arecent study demonstrated that bupropion was equally effective forsmoking cessation independent of a past history of major depression,suggesting that this therapeutic effect is independent of, or notexclusive to, its antidepressant properties (Hayford et al. 1999).Furthermore, the initial trials assessing bupropion's effectiveness insmoking cessation excluded depressed patients (e.g. Jorenby et al.1999). Moreover, other antidepressants, including the selectiveserotonin reuptake inhibitors, with the possible exception of thetricyclic nortryptiline, have been shown to be rather ineffective inreducing quit rates in non-depressed smokers (see Kotlyar et al. 2001).Therefore, there may be unique neurochemical and behavioral sequelae ofbupropion administration, beyond that of its antidepressant propertiesthat engender its antismoking efficacy.

Reward deficits associated with withdrawal from drugs of abuse can beused as an animal model to measure symptoms of “diminished interest orpleasure” that characterizes both drug-withdrawal-induced andnondrug-induced depressions with construct, convergent, and predictivevalidity (Geyer and Markou 1995; Barr et al. 2002; Cryan et al. 2002;2003). The use of intracranial self-stimulation (ICSS) has providedinvestigators with a reliable behavioral readout to assess suchalterations in brain reward after cessation of drug administration (i.e.drug withdrawal) (Leith and Barrett 1976; Markou and Koob 1991; EppingJordan et al. 1998; Cryan et al. 2002; Spielewoy and Markou 2002). Usingthis paradigm, we have previously shown that withdrawal from chronicnicotine induces a dramatic decrease in brain reward function asreflected by elevations in brain reward thresholds (Epping-Jordan et al.1998; Harrison et al. 2001 a; Semenova and Markou 2003). This thresholdelevation is one of the few operational measures of the affectiveaspects of the nicotine withdrawal syndrome (Kenny and Markou 2001). Inaddition to the affective symptoms, nicotine withdrawal in humans isalso characterized by somatic symptoms such as bradycardia, insomnia,gastrointestinal discomfort, and increased appetite (Hughes et al.1991). These symptoms are amenable to visual assessment in withdrawinganimals (Malin et al. 1992; Malin 2001). A clear dissociation has beendemonstrated recently between the neuroanatomical sites of elevations inbrain stimulation reward thresholds that are mediated through centralmechanisms, and those somatic withdrawal signs that appear to be bothcentrally and peripherally mediated (Hildebrand et al. 1999; Watkins etal. 2000). It has been postulated that affective signs are of greatmotivational significance in contributing to relapse and continuednicotine use (Markou et al. 1998). Further, somatic signs of nicotinewithdrawal also may contribute to smoking behavior. Thus, animal modelsof the affective and somatic aspects of nicotine withdrawal areimportant tools for understanding the neurobiological bases of nicotinedependence and for developing effective treatment strategies tofacilitate nicotine abstinence (Kenny and Markou 2001; Malin 2001). Thepresent studies investigated the effects of bupropion on brain rewardfunction under baseline conditions and subsequent to withdrawal fromchronic nicotine. In addition, the effects of bupropion on the somaticsigns of withdrawal were assessed.

Materials and Methods

Animals

Male Wistar rats (Charles River, Raleigh-Durham, N.C., USA), 275-350 gupon arrival were housed in pairs with food and water available adlibitum, except during testing, in a temperature and humidity controlledvivarium (21° C.). Rats were maintained on a 12-h reverse light/darkcycle with lights on at 1800 hours. All experimental procedures occurredduring the dark cycle, and in accordance with the Institutional AnimalCare and Use Committee of The Scripps Research Institute. Animals wereallowed to habituate to their new environment for at least one weekbefore the start of any procedure, during which time they were handledat least twice.

ICSS apparatus

All training and testing occurred in 16 Plexiglas test chambers(25×31×24 cm; Med Associates, Georgia, Vt., USA) separately housed inlarger sound-attenuating boxes (San Diego Instruments, San Diego,Calif., USA). Centered on a side wall of each operant chamber was ametal wheel manipulandum (5 cm wide) requiring approximately 0.2 N forceto rotate it a quarter turn. Brain stimulation was delivered usingconstant current stimulators (Stimtech model 1200; San DiegoInstruments). Subjects were connected to the stimulation circuit viabipolar leads (Plastics One, Roanoke, Va., USA) attached to gold-contactswivel commutators (model SL2C; Plastics One) that were mounted abovethe chamber. Stimulation parameters, data collection and all testsession functions were controlled by a microcomputer.

Surgery

ICSS Electrode Implantation

When the subjects reached a minimal weight of 325 g, stainless steelbipolar electrodes, with a diameter of 0.25 mm (model MS303/2; PlasticsOne) cut to 11 mm in length, were implanted into the medial forebrainbundle at the level of the posterior lateral 348 hypothalamus. Briefelectrical stimulation of this region is reinforcing as indicated by thefact that rats perform an operant (turn a wheel) to receive theelectrical stimuli (Markou and Koob 1992). A discrete-trial ICSSprocedure was used that provides one with current-intensitiesthresholds, a measure of reward (Markou and Koob 1992). Many drugs ofabuse increase brain reward function, as indicated by a decrease in ICSSthreshold, whereas withdrawal from drugs of abuse decreases brain rewardas indicated by elevations in thresholds.

Subjects were anesthetized with an isoflurane/oxygen vapor mixture (1-3%isoflurane) and secured in a stereotaxic frame (David Kopf Instruments,Tujunga, Calif., USA) with the incisor bar elevated 5.0 mm above theinteraural line. Electrodes were implanted according to the followingcoordinates: AP −0.5 mm from bregma; ML±1.7 mm; and DV −8.3 mm fromdura. Half of the subjects received electrodes on the right side of thebrain, and half on the left to counterbalance for possible brainasymmetries. Dental acrylic (Teets methyl methacrylate denture material;CoOral-Lite Mfg. Co., Diamond Springs, Calif., USA) anchored theelectrode to four stainless steel screws embedded in the skull. Thesurgical wound was flushed with a solution of 13.3 mg/ml gentamicinsulfate dissolved in physiological saline, closed with silk sutures, andcovered with a povidone-iodine antiseptic ointment. After surgery,animals were allowed to recover at least 7 days before the start ofbehavioral training.

Osmotic Pump Implantation and Removal

Subjects were anesthetized with an isoflurane/oxygen vapor mixture (1-3%isoflurane) and prepared with subcutaneous osmotic minipumps [Alzetmodel 2ML1 (7 days); Alza Corporation, Palo Alto, Calif., USA] along theback, parallel to the spine, with the flow meter directed posteriorly.Pumps were filled with either physiological saline or nicotine solution.The concentration of the latter was adjusted according to animal weightand pumping rate to deliver a dose of 3.16 mg/kg per day (9 mg/kg perday nicotine hydrogen tartrate). The wound was closed with stainlesssteel wound clips and covered with a povidone-iodine antisepticointment. Pumps were surgically removed seven days later underisoflurane anesthesia, the wounds re-clipped and treated with theantiseptic ointment.

Brain Stimulation Reward Threshold Procedure (ICSS)

Subjects were initially trained to turn the wheel manipulandum on afixed ratio 1 (FR1) schedule of reinforcement. For each quarter turn,subjects received a 500 ms train of 0.1 ms cathodal square wave pulsesat a frequency of 100 Hz. After successful acquisition of the response,defined as 200 consecutive reinforcements within allotted time period(usually <20 min) subjects were trained on a modification of theKornetsky and Esposito discrete-trial currentthreshold procedure(Kornetsky and Esposito 1979), as described previously by Markou andKoob (1992).

Trials began with an electrical stimulus followed by a 7.5-s responsewindow within which the animal could make a response to receive a secondcontingent stimulus identical to the initial noncontingent stimulus. Aresponse during this 7.5-s window was considered a positive response,whereas the lack of a response was considered a negative response.Additional responses during a 2-s period immediately following apositive response had no consequence. The intertrial interval (ITI) thatfollowed either a positive response or the end of the response window(in the case of a negative response) ranged from 7.5 to 12.5 s with anaverage duration of 10 s. Responses that occurred during the ITIresulted in a further 12.5 s delay before the onset of the subsequenttrial. During training on the discrete-trial procedure, the duration ofthe ITI and time-out response delays were gradually increased until theanimals performed consistently at a fixed stimulation intensity.Subjects then were tested on a current-threshold procedure in whichstimulus intensities were varied during four alternating descending andascending series with a step size of 5 mA. Blocks of three trials wereoffered at each current step with the starting current of the firstdescending series set at 30-40 mA above the subjects' individualbaseline thresholds estimated at the end of preliminary training. Adescending series was terminated after two consecutive blocks of trialsduring which the animal failed to make a positive response on at leasttwo out of the three trials or after 15 successive decrements werepresented. An ascending series was terminated after two consecutiveblocks of trials during which the animal made positive responses on atleast two out of the three trials or after 15 successive increments werepresented. The mean of the four current thresholds from the alternatingseries was defined as the subject's ICSS threshold for that session. Aseries was terminated after either 15 stimulus increments (ordecrements) had occurred, or after the determination of the thresholdfor the series (see below). Each test session typically lasted 30 min.

Thresholds

The current threshold for each descending series was defined as thestimulus intensity between the successful completion of a set of trials(positive responses during two or more of the three trials) and thestimulus intensity for the first set of trials, of two consecutive sets,during which the animal failed to respond positively on two or more ofthe three trials. During the ascending series, the threshold was definedas the stimulus intensity between the unsuccessful completion of a setof trials (negative responses during two or more of the three trials)and the stimulus intensity for the first set of trials, of twoconsecutive sets, during which the animal responded positively on two ormore of the trials. Thus, during each test session, four thresholds weredetermined and the mean of these values was taken as the threshold foreach subject. After training in the above-mentioned brain stimulationreward procedure, rats were tested until stable baseline thresholds hadbeen achieved (±10% over a 5-day period). Drug testing was initiatedonly after performance had stabilized, which typically occurred after2-3 weeks of baseline testing. Return to baseline threshold levels wasrequired between drug injections.

Ratings of Somatic Signs of the Withdrawal Syndrome

Rats were placed individually in transparent plastic cylindricalcontainers (30×38 cm) in which they could move freely. Subjects werehabituated to the containers for 10 min each day over 3 days before thefirst test session. During the test sessions the rats were observedblindly by an experienced observer for 10 min and the frequency of thefollowing signs was recorded based on a checklist of nicotine abstinencesigns (Hildebrand et al. 1999; Watkins et al. 2000; Malin 2001): bodyshakes, chews, cheek tremors, escape attempts, eye blinks, foot licks,gasps, genital licks, head shakes, ptosis, scratches, teeth chattering,writhes and yawns. Multiple successive counts of any sign required adistinct pause between episodes. Ptosis, if present continuously, wasonly counted once per minute. For statistical analyses, the total numberof somatic signs was defined as the sum of individual occurrences of theabove mentioned withdrawal signs. Further, the categories of “abdominalconstrictions” included gasps and writhes; “facial fasciculation”included cheek tremors, chews, and teeth chattering; and “miscellaneousother signs” included shakes, escape attempts, licks, scratches, andyawns.

Drugs

(−)-Nicotine hydrogen tartrate salt (Sigma, St Louis, Mo., USA) wasdissolved in sterile physiological saline (0.9% sodium chloride).Bupropion hydrochloride generously provided by Glaxo-SmithKline,Research Triangle Park, N.C., USA) was dissolved in sterile distilledwater. Nicotine doses are expressed as the free base, while bupropiondoses are expressed as the salt. All injections were administered in avolume of 1 mvkg.

Experiment 1: Effects of Acute Bupropion Treatment on ICSS RewardThresholds under Baseline Conditions

Bupropion (0, 10 20, 30 mg/kg, IP, n=8) was administered (30 minpretreatment) according to a within-subjects Latin square design, with aminimum of 3 days between consecutive drug treatments. After return tobaseline values, all animals were given an injection of bupropion (40mg/kg, IP), followed by another injection of 60 mg/kg bupropion IP atleast 3 days later. These two highest bupropion doses were administeredlast to avoid any potential longlasting effects of high bupropion doses.Nevertheless, no such effects were observed. Although the doses used inthese studies are higher than the recommended daily dose in humans (300mg/day) [see GlaxoSmithKline product information on Zyban (bupropionhydrochloride) sustained release tablets 2002, available from on theinternet at us.gsk.com/products/assets/us_zyban.pdf, such comparisonsare at best rough because of differences in pharmacokinetics andmetabolism of drugs in different species. It should be noted that thedoses of bupropion used are in the range that produce robustantidepressant-like behavioural effects in rodents (e.g. Cryan et al.2001) and that increase hypothalamic concentrations of extracellulardopamine and norepinephrine (Li et al. 2002).

Experiment 2: Effects of Acute Bupropion on Acute Nicotine-InducedLowering of ICSS Thresholds

In a separate naive group of animals bupropion (0, 5, 10, 20 mg/kg, IP,n=10) was administered (30 min pretreatment) in combination withnicotine (0, 0.25 mg/kg, SC; 15 min pretreatment) according to awithin-subjects Latin square design, with a minimum of 3 days betweenconsecutive drug treatments. The dose of nicotine was selected fromprevious dose-response studies from our laboratory showing maximalrewarding effects of acute nicotine at this dose (Harrision et al.2002), and the dose of bupropion was selected based on the results fromexperiment 1 described above.

Experiment 3: Effects of Acute Bupropion Treatment on ICSS RewardThresholds Subsequent to Withdrawal from Chronic Nicotine or SalineAdministration

Naive animals were prepared with osmotic minipumps containing either anindividualized nicotine solution (3.16 mg/kg per day, free base) orsaline. The dose of nicotine was selected based on extensive previousdose-response studies which showed that exposure to this dose for 7 daysresults in a robust and reproducible withdrawal syndrome as assessed byboth ICSS (elevations in brain reward thresholds) and somatic signs ofwithdrawal (Epping-Jordan et al. 1998; Harrison et al. 2001 a; Malin2001). This dose maintains stable plasma nicotine levels (44 ng/ml)comparable to those reported for smokers consuming 30 cigarettes daily(4042 ng/ml) (see Epping-Jordan et al. 1998). After implantation of theminipumps, thresholds were assessed daily thereafter to assess theeffects of chronic nicotine exposure on reward thresholds. Seven days(exactly seven 24-h periods) after pump insertion, the pumps wereremoved in order to precipitate the nicotine withdrawal syndrome. ICSSthresholds were measured 12, 18, 24, 36, 48 and 72 h after pump removal.Animals were injected with bupropion (0, 10, 20, 40 mg/kg IP; n=8-11) 30min prior to the 18-h time point. This time point was selected becauseit is the time point when maximal withdrawal-induced deficits(elevations in brain reward thresholds) were observed in previousexperiments (Epping-Jordan et al. 1998; Harrison et al. 2001 a; Semenovaand Markou 2003).

Experiment 4: Effects of Acute Bupropion Treatment on Somatic SignsSubsequent to Withdrawal from Chronic Nicotine Administration

Separate drug-naive rats were prepared with osmotic minipumps containingnicotine (3.16 mg/kg per day for 7 days) dissolved in saline. As statedabove, this dose of nicotine was selected based on extensivedose-response studies and is one which results in a robust withdrawalsyndrome in the rat (Malin et al. 1992; Epping-Jordan et al. 1998;Harrison et al. 2001 a; Malin 2001; Skjei and Markou 2003). Further,this nicotine dose was selected to allow direct comparison of theeffects of bupropion on somatic signs (present experiment 4) and onreward threshold elevations (experiment 3 described above) observedduring nicotine withdrawal. To induce the somatic signs of nicotinewithdrawal the minipumps were removed 6 days and 18 h afterimplantation. The behavioral observations were conducted 6, 12 and 24 hafter the removal of the minipumps. The rats were injected withbupropion (0, 5, 10 20, 40 mg/kg, IP; n=6-8), 30 min prior to the 12-htime point. The selection of the time points was based on thetime-course of the expression of the somatic signs after the terminationof nicotine exposure (Epping-Jordan et al. 1998; Harrison et al. 2001 a;Semenova and Markou 2003; Skjei and Markou 2003).

Data Analyses and Statistics

Threshold data were expressed as a percentage of the previous three to5-day baseline values before each drug manipulation or the implantationof the minipump. Percent values are used for the statistical analyses ofthreshold data because raw current intensity threshold values ofindividual rats vary during baseline conditions due to minor variationsin electrode placement and other poorly understood individual subjectfactors. Extensive previous work in our laboratory has indicated thatthe use of within-subject designs as the ones described in thismanuscript and the expression of threshold data as a percentage ofstable baseline thresholds before the implementation of any manipulationis a reliable and validated method for assessing changes in brain rewardfunction (see Markou and Koob 1992). All data were analyzed using theappropriate within-subject, and mixed-design ANOVAs. Statisticallysignificant effects were followed where appropriate with Fisher'sindividual comparison tests. The level of significance was set atP<0.05.

Results

Experiment 1: Effects of Acute Bupropion Treatment on ICSS CurrentThresholds under Baseline Conditions

Acute bupropion treatment induced a pronounced lowering of brain rewardthresholds [F(5, 35)=7.445, P<0.001] which is indicative of an increasein brain reward function (FIG. 23). Post-hoc analyses indicated thatbupropion at all doses tested and in a dose-dependent fashion up to 40mg/ kg (40 mg/kg was significantly different from 10 mg/kgbupropion-treated animals) lowered brain reward thresholds compared tothe vehicle conditions. Raw mean 3-day baseline thresholds were in therange of 128.19-143.63 mA.

Experiment 2: Effects of Acute Bupropion on Acute Nicotine-InducedLowering of ICSS Thresholds

As in experiment 1, bupropion (10 and 20 mg/kg, but not 5 mg/kg) loweredbrain reward thresholds in animals that were co-treated with saline[F(3,54)=7.822, P<0.001] (FIG. 24). Similarly, nicotine treatmentresulted in lowering of brain reward thresholds [F(1,18)=11.550,P<0.003], which is consistent with previous findings (Harrison et al.2002; Skjei and Markou 2003). ANOVA revealed a strong trend towards asignificant interaction between the factors nicotine and bupropion [F(3,54)=2.405, P=0.077]. Planned comparisons indicated that bupropion at adose that was ineffective on its own (5 mg/kg) completely reversed thereward-enhancing effects of acute nicotine. However, higher bupropiondoses (10 and 20 mg/kg) in combination with nicotine resulted inthreshold lowerings that were not significantly different from thresholdlowerings induced by nicotine administration alone. Raw mean 3-daybaseline thresholds were in the range of 111-133.26 mA.

Experiment 3: Effects of Acute Bupropion Treatment on ICSS CurrentThresholds During and Subsequent to Withdrawal from Chronic Nicotine orSaline Administration

Administration of nicotine via minipumps resulted in a small yetsignificant reduction in brain reward threshold [F(1,74)=5.75, P<0.05],with the maximal effect seen on day 3 of administration. However, thethreshold values returned to baseline levels (P>0.8) and were notdifferent from those of saline-treated animals at the time of pumpremoval on day 7 (P>0.1). This pattern of results suggests that therewas tolerance development to the mild threshold-lowering effects ofnicotine (see FIG. 25). Raw mean 5-day baseline values before theimplantation of the pumps were 119.75 mA for saline-treated animals and120.08 mA for animals prepared with nicotine-containing minipumps.

To assess the effects of nicotine withdrawal on brain reward thresholds,thresholds after the removal of the minipumps were expressed as percentof the 5-day baseline threshold values assessed under baselineconditions before the implantation of the minipump. Thus, these percentchanges reported for the withdrawal period reflect changes from baselinebrain reward thresholds and not changes from thresholds during nicotineexposure when thresholds could have been altered under the influence ofnicotine. Nevertheless, as described above, thresholds ofnicotine-treated rats were back to baseline threshold levels by day 7 ofexposure to nicotine. As anticipated, withdrawal from chronic nicotineadministration significantly elevated brain reward thresholds[F(1,68)=22.219, P<0.001] compared to thresholds of saline-pretreatedanimals. In addition, the ANOVA revealed that there was a significantchange in thresholds over time [F(5,340)=23.047, P<0.001]. Further,there were significant interactions between the factors pretreatment(nicotine or vehicle) and time [F(5, 340)=5.731, P<0.001], and thefactors treatment (bupropion or vehicle) and time [F(15,340)=13.568,P<0.001].

Planned comparisons revealed that nicotine withdrawal resulted in asignificant elevation in reward thresholds in saline-injected animals atall time points tested up to 36 h post-withdrawal. At the 48- and 72-htime points, the withdrawal-induced deficits had normalized. Thirtyminutes following administration of bupropion (17.5 h post withdrawal)there was a significant and dose-dependent lowering of reward thresholdsat all doses, regardless of whether the animals had been pretreated withnicotine or not. Furthermore, following injection of bupropion (40mg/kg) there was no longer any significant difference between animalsthat had been pretreated with saline and those pretreated with nicotine.Moreover, in saline pretreated animals, this reduction in threshold wasshort-lived and completely reversed at the next time point tested (24 h)in all animals, which was similar to the results of experiment 1.Interestingly, however, animals pretreated with nicotine and injectedwith 40 mg/kg bupropion still exhibited a significant (P<0.001)reduction in reward thresholds compared to nicotine-pretreated animalsthat were injected with saline (see FIG. 26A).

Analysis of the area under the curve of the threshold data (over theentire 72-h withdrawal period) demonstrated a significant effect ofnicotine pretreatment [F(1,68)=13.705, P<0.001]. There was no overallstatistically significant effect of drug (bupropion) treatment, or adrug×pump interaction. Planned comparisons indicated that animalspretreated with nicotine had a higher area under the curve thresholdlevel than corresponding animals pretreated with saline. This elevationwas completely reversed in animals treated with the highest dose ofbupropion (40 mg/kg) tested (see FIG. 26B). Raw mean 5-day baselinethresholds were in the range of 108.69- 131.83 mA.

Experiment 4: Effects of Acute Bupropion Treatment on Somatic SignsSubsequent to Withdrawal from Chronic Nicotine Administration

Six hours after removal of the nicotine-containing minipumps and priorto any drug manipulation, there was a significant increase in the amountof total somatic signs of abstinence. These signs were also evident tothe same extent 12 h following withdrawal in nicotineexposed rats thatwere treated with vehicle before the 12-h observation time point. Therewas a significant interaction of bupropion treatment with time[F(4,31)=23.76 P<0.001]. Post hoc comparisons revealed that bupropion(20-40 mg/kg) administered 30 min prior to the 12-h withdrawal timepointresulted in a reversal of the expression of total somatic signs comparedto the number of somatic signs exhibited by the same animals at the 6-htime point before the bupropion administration (withinsubjectscomparison) and those of vehicle-treated rats that also were undergoingnicotine withdrawal (betweensubjects comparison) (see FIG. 27A).Finally, it should be noted that there were no significant differencesbetween the groups' total number of somatic signs before theadministration of bupropion (6 h post-pump removal; see FIG. 27A) orseveral hours post-treatment (24 h post pump removal and 12.5 hpostbupropion administration; P>0.05; vehicle=20.7±7.3; bupropion 5mg/kg=18.6±4; bupropion 10 mg/kg=17.1±4.6; bupropion 20 mg/kg=14.1±2.2;bupropion 40 mg/ kg=14.6±2.6). These data indicate that the differencebetween the saline and bupropion-treated rats (12 h postpump removal) isnot due to baseline differences, and that acute treatment with bupropionhas no long-term (12.5 h) effect on the expression of the somatic signsof nicotine withdrawal.

One-way ANOVAs were used to examine the effects of bupropion on theindividual somatic signs at the 12-h withdrawal time point. It wasdemonstrated that there was a significant decrease in the amount ofabdominal constrictions (writhes and gasps) with bupropionadministration [F(4,31)=4.037 P<0.01], but not on other signs. Plannedcomparison revealed that bupropion (10-40 mg/ kg) reversed thewithdrawal-induced increase in abdominal constrictions (see FIG. 27B).

Discussion

The present studies provide striking evidence that bupropion acts toalter brain reward circuits influenced by nicotine. First, bupropionincreases brain reward function under baseline conditions. Second, atlow doses it blocks the reward facilitating effects induced by acutenicotine. Third, it reverses both the negative affective aspects ofnicotine withdrawal that are expressed as reward deficits and thesomatic signs of withdrawal. The ability of bupropion to increase brainreward function under baseline conditions, as indicated by lowering ofICSS thresholds, is in stark contrast to data that we and otherspreviously generated with other antidepressants using ICSS. Such studiesshowed that acute administration of antidepressants such as desipramine,paroxetine or fluoxetine had no effect or induced elevations inthresholds under baseline conditions (Atrens et al. 1977; Katz andCarroll 1977; Binks et al. 1979; Hall et al. 1990; Markou et al. 1992;Lee and Kornetsky 1998; Lin et al. 1999; Harrison et al. 2001 a; 2001b;Cryan and Markou, unpublished observations). This differential effect ofbupropion may underlie its superiority over many other antidepressantsin smoking cessation. In contrast, Mc-Carter and Kokkinidis (1988)demonstrated that 28-day treatment with bupropion had no effect on therate of responding for ICSS. It is difficult to draw comparisons betweenthese two data sets, however, because the

McCarter and Kokkinidis procedure is operationally different from oursand used only one bupropion dose (20 mg/kg). It has been suggested,given the high incidence of smoking among psychiatric populationsincluding depressed patients (Leonard et al. 2001), that smokers may beusing nicotine to self-medicate depressive symptomatology (Glassman etal. 1990; Markou et al. 1998; Markou and Kenny 2002). Indeed, theselargely epidemiological observations have recently been confirmedneurochemically, where it was shown that locus coeruleus a2noradrenergic receptors are down-regulated in smokers to a similarextent as previously reported with antidepressant medications (Klimecket al. 2001). Furthermore, withdrawal from nicotine results in deficitsin brain reward function similar to those observed in major affectivedisorders (Markou and Kenny 2002). Taken together, these observationssuggest that antidepressant medications may be an effective treatment indecreasing smoking rates, because they treat the underlying depressivesymptomatology. However, there is growing evidence that antidepressants,such as the selective serotonin reuptake inhibitors, may reduce nicotineintake only, if at all, in certain subsets of depressed patients and mayhave limited efficacy in non-depressed patients (Kotlyar et al. 2001).In contrast, bupropion is equally effective in healthy and psychiatricpopulations and therefore its efficacy extends beyond that of itsantidepressant properties (Hayford et al. 1999). We suggest here thatthe antismoking efficacy of bupropion may be due to its additionaleffects on brain reward function even under baseline non-withdrawalconditions, effects not shared by other antidepressants.

The neurochemical mechanisms underlying bupropion's actions are stillnot well elucidated. Recent data indicate that bupropion's behaviouraleffects may be due to its effects on the noradrenergic system (Ascher etal. 1995; Cryan et al. 2001; Dong and Blier 2001). Nonetheless, unlikemany antidepressants it also acts as a dopamine reuptake inhibitor(albeit in the micromolar range) (Ascher et al. 1995) and microdialysisstudies have shown that acute administration of bupropion in the doserange that decreases brain reward thresholds in the current studies,increases extracellular dopamine (Nomikos et al. 1989; Li et al. 2002).Given that the dopamine system long has been associated with brainreward mechanisms, this property of bupropion may contribute to itsrewardenhancing effects. Dopaminergic mechanisms also have beenimplicated in both the rewarding aspects of nicotine and in themanifestation of the nicotine withdrawal syndrome (Hildebrand etal.,1998, 1999; Kenny and Markou 2001; Ferrari et al. 2002; Mansvelderet al. 2002;

Picciotto and Corrigall 2002). However, recent studies have shown thatwhereas increases in nucleus accumbens dopamine concentrations areessential for ICSS responding to occur, they do not parallel thetemporal dynamics of ICSS (Garris et al. 1999). Thus, whilst dopamine isinvolved in certain aspects of reward (most likely novelty orexpectancy), other reciprocal neurochemical mechanisms are recruited tosustain the rewarding “hedonic” aspects of self-stimulation (Wise andStein 1969; Herberg et al. 1976; Garris et al. 1999). Thus, by usingICSS at the level of the lateral hypothalamus we are able to obtain anindex of trans-synaptic modulation of activity in limbic areas, such asthe nucleus accumbens, an area strongly but not exclusively implicatedin the mediation of reward, thus allowing the detection ofnon-dopaminergic neurotransmitter modulation. Such modulation isrelevant to the current studies because bupropion has effects at bothdopaminergic and noradrenergic transporters. Indeed, recentelectrophysiological data suggest that sustained bupropion treatment(for 2 days by minipump) altered the firing of locus coereleusnoradrenergic neurons and to a lesser extent serotonergic raphe neuronsbut not dopaminergic ventral tegmental area neurons (Dong and Blier2001). Furthermore, a recent imaging study in humans demonstrated thatat therapeutically effective doses bupropion has only 22% occupancy atthe dopamine transporter (Meyer et al. 2002), indicating that theefficacy of bupropion is probably not solely dopamine-mediated.

Moreover, recent evidence suggests that bupropion may act as afunctional antagonist at neuronal nicotinic acetylcholine receptors(Fryer and Lukas 2002; Slemmer et al. 2000; Miller et al. 2002). Ourdata showing blockade of the acute rewarding effects of nicotine bybupropion at a dose that was ineffective on its own supports thishypothesis. At higher doses bupropion's independent effects infacilitating brain reward function are manifested, making it difficultto disentangle its potential nicotinic antagonist effects. Nonetheless,our observations also give neurochemical credence to the clinicalpractice of initiating bupropion therapy prior to nicotine cessation(Hughes et al. 1999). It is likely that bupropion may act acutely toattenuate the rewarding effects of nicotine, thus increasing thelikelihood of cessation. Indeed, mecamylamine, a classical nicotinicreceptor antagonist, has been shown to be effective as a smokingcessation aid when used in combination with a nicotine patch (Rose etal. 1994). Interestingly, bupropion generalizes to nicotine in drugdiscrimination paradigms. However, unlike nicotine's discriminatoryproperties, those of bupropion are insensitive to blockade bymecamylamine, indicating a differential mechanism of action mediatingthe discriminatory cues of bupropion and nicotine (Young and Glennon2002; Wiley et al. 2002).

The reversal of the affective aspects of withdrawal by all doses ofbupropion tested here (10, 20 and 40 mg/kg) further suggests that itseffects on reward systems are relevant to its therapeutic efficacy.Further, our data show clear differential effects of bupropion on rewardthresholds in nicotine-pretreated animals compared with salinetreatedcontrols. In the case of the latter, bupropion induced a short-lastinglowering of thresholds that returned to baseline at the next time pointtested, which is consistent with the results of our initial dose-findingstudies. Animals that had been pretreated with chronic nicotine, whilstshowing minor reductions in thresholds during continuous nicotineexposure exhibited marked elevations in thresholds upon cessation ofnicotine administration, as shown previously (Epping-Jordan et al.1998). The highest dose of bupropion tested (40 mg/kg) exhibited aprotracted reversal of these withdrawal deficits, with thresholds stillsignificantly lower than those of vehicle-challenged animals previouslytreated with nicotine at the second post-injection testing period (6.5hours post-bupropion). Furthermore, an analysis of reward thresholdsover the entire 72-h period indicates that there was no difference inanimals undergoing nicotine withdrawal and injected with bupropion (40mg/ kg) and those not undergoing withdrawal. Administration of lowerbupropion doses (10 and 20 mg/kg) also reversed the reward deficits,although this effect was not as protracted as that seen afteradministration of 40 mg/kg bupropion. These findings indicate thatbupropion induces reversal of a withdrawal deficit that is differentialfrom its effects on reward function under baseline conditions. It islikely that this prolongation of the attenuation of withdrawal-inducedreward deficits is of prime importance to the anti-smoking properties ofbupropion, as the affective aspects of withdrawal are considered to be acritical factor in motivating further drug use (Kenny and Markou 2001).These data are in contrast to previous data with the antidepressantsfluoxetine and paroxetine which did not much alter the negativeaffective aspects of withdrawal syndrome when given alone (Harrison etal 2001 a, 2001 b). However, such deficits in reward function werecounteracted when a selective serotonin reuptake inhibitor was given incombination with a serotonin1A autoreceptor antagonist (Harrison et al2001 a; 2001 b). These findings are consistent with recent datademonstrating a role for the serotonergic dorsal raphe nucleus inmediating some of the affective aspects of nicotine withdrawal (Cheetaet al. 2000). Further, Rasmussen and colleagues (1997; 2000) have shownthat 5-HT1A receptor antagonists reversed the increases in the auditorystartle reflex observed during nicotine withdrawal in rats.

In addition to counteracting the affective components of nicotinewithdrawal, bupropion also reversed the expression of the somaticaspects of the spontaneous nicotine withdrawal syndrome. Thisobservation is consistent with a preliminary study reporting thatbupropion reversed somatic signs of nicotine withdrawal (Malin 2001).The mechanisms underlying this reversal are not clear. Because reduceddopaminergic signaling is thought to be a key factor in themanifestation of somatic signs of nicotine withdrawal (Hildebrand et al.1999), it is probable that bupropion's direct effects on the dopaminesystem may counteract this deficit. However, bupropion's effects onother pathways such as the noradrenergic and serotonergic systems cannotbe excluded, as they also have been implicated in mediating theexpression of somatic signs (Kenny and Markou 2001; Malin 2001). Thereversal of both the affective and the somatic aspects of nicotinewithdrawal also may explicate the marked efficacy of bupropion insmoking cessation, as the negative aspects of withdrawal are postulatedto contribute to the tobacco smoking habit (Glassman et al. 1990;Breslau et al. 1992; Laje et al. 2001). Indeed, recent clinical studiesdemonstrated that negative affect was a significant mediator ofbupropion's effects on smoking cessation (Shiffman et al. 2000; Lermanet al. 2002).

It should be noted that as all of the effects of bupropion in thepresent studies were investigated following acute administration, it isnot known yet whether such effects persist with chronic administration.The only previous study on bupropion's effects on brain reward functionwas with chronic administration under baseline conditions (McCarter andKokkinidis 1988). This study demonstrated that 28-day treatment withbupropion had no effect on the rate of responding for ICSS. It isdifficult to draw comparisons between these two data sets, however,because McCarter and Kokkinidis investigated the effects of bupropiononly under baseline conditions and their procedure is operationallydifferent from ours. The fact that only one bupropion dose (20 mg/kg)was used in their studies, and that no acute testing was carried outmakes it more difficult to assess whether such negative effects arerelevant to the present data. One of the major drawbacks of using brainreward thresholds and somatic signs of nicotine withdrawal in the rat asindices of similar effects in humans is that these aspects of withdrawalare relatively short-lasting, in the range of 2-3 days (Kenny and Markou2001). Thus, this short duration makes investigating the effects ofchronic administration of potential therapies more difficult.Nevertheless, this window of 2-3 days may be sufficient to allow thedetection of potential therapeutic effects of novel treatments,similarly to models of other psychiatric disorders (e.g. Geyer et al.2001; Lucki 2001; Cryan et al. 2002; Seong et al. 2002), and thusfurnish the model with predictive validity (Geyer and Markou 1995).Further studies are warranted to clarify these issues and to assess theeffects of chronic bupropion administration on the various aspects ofnicotine withdrawal.

In conclusion, these data demonstrate that the utility of bupropion asan aid to smoking cessation may be due to its ability to alter therewarding aspects of acute nicotine and to reverse the negativeaffective and somatic aspects of the nicotine withdrawal syndrome. Thebupropioninduced increases in brain reward function even undernon-nicotine withdrawal conditions also may contribute to a shift in themotivational priorities of smokers, and thus lead to decreases intobacco consumption. Further studies will be needed to assess theeffects of chronic bupropion treatment on brain reward and nicotinewithdrawal.

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While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1. A method for treating a metabotropic glutamate disorder, comprisingadministering to a subject in need thereof, an effective amount of atleast one antagonist which modulates metabotropic glutamate receptor 2,metabotropic glutamate receptor 3, and metabotropic glutamate receptor5, thereby treating the disorder.
 2. A method for treating ametabotropic glutamate disorder, comprising administering to a subjectin need thereof, an effective amount of at least one antagonist whichmodulates metabotropic glutamate receptor 2, and metabotropic glutamatereceptor 5, thereby treating the disorder.
 3. A method for treating ametabotropic glutamate disorder, comprising administering to a subjectin need thereof an effective amount of at least one antagonist whichmodulates metabotropic glutamate receptor 3 and metabotropic glutamatereceptor 5, thereby treating the disorder.
 4. The method of claim 1,wherein the disorder is an addictive disorder.
 5. The method of claim 4,wherein the addictive disorder is nicotine addiction, alcohol addiction,opiate addiction, amphetamine addiction, methamphetamine addiction, orcocaine addiction.
 6. The method of claim 4, wherein the addictivedisorder is nicotine addiction.
 7. The method of claim 4, wherein theaddictive disorder is cocaine addiction.
 8. The method of claim 1,wherein the disorder is depression.
 9. The method according to claim 1,wherein the antagonist is 2-methyl-6-(phenylethynyl)-pyridine.
 10. Acombination comprising (a) at least one active ingredient selected froma metabotropic glutamate receptor 2 antagonist and a metabotropicglutamate receptor 3 antagonist, and (b) at least one metabotropicglutamate receptor 5 antagonist, in which the active ingredients arepresent in each case in free form or in the form of a pharmaceuticallyacceptable salt, and optionally at least one pharmaceutically acceptablecarrier; for simultaneous, separate or sequential use.
 11. A combinationcomprising (a) at least one active ingredient which exhibitsantagonistic activity against the metabotropic glutamate receptor 2 andthe metabotropic glutamate receptor 3, and (b) at least one metabotropicglutamate receptor 5 antagonist, in which the active ingredients arepresent in each case in free form or in the form of a pharmaceuticallyacceptable salt, and optionally at least one pharmaceutically acceptablecarrier; for simultaneous, separate or sequential use.
 12. A combinationcomprising (a) at least one metabotropic glutamate receptor 2antagonist, and (b) at least one active ingredient which exhibitsantagonistic activity against the metabotropic glutamate receptor 3 andthe metabotropic glutamate receptor 5, in which the active ingredientsare present in each case in free form or in the form of apharmaceutically acceptable salt, and optionally at least onepharmaceutically acceptable carrier for simultaneous, separate orsequential use.
 13. A combination comprising (a) at least onemetabotropic glutamate receptor 3 antagonist, and (b) at least oneactive ingredient which exhibits antagonistic activity against themetabotropic glutamate receptor 2 and the metabotropic glutamatereceptor 5, in which the active ingredients are present in each case infree form or in the form of a pharmaceutically acceptable salt, andoptionally at least one pharmaceutically acceptable carrier; forsimultaneous, separate or sequential use.
 14. The combination accordingto claim 10 which is a combined preparation or a pharmaceuticalcomposition.
 15. The combination according to claim 10 for simultaneous,separate or sequential use in the treatment of an addictive disorder ordepression.
 16. A method of treating a warm-blooded animal having anaddictive disorder or depression comprising administering to the animala combination according to claim 10 in a quantity which is jointlytherapeutically effective against an addictive disorder or depressionand in which the compounds can also be present in the form of theirpharmaceutically acceptable salts.
 17. A pharmaceutical compositioncomprising a quantity, which is jointly therapeutically effectiveagainst an addictive disorder or depression, of a pharmaceuticalcombination according to claim 10 and at least one pharmaceuticallyacceptable carrier.
 18. A use of a combination according to claim 10 forthe preparation of a medicament for the treatment of an addictivedisorder or depression.
 19. A commercial package comprising acombination according to claim 10 together with instructions forsimultaneous, separate or sequential use thereof in the treatment of anaddictive disorder or depression.
 20. A method for treating substanceabuse, comprising administering to a subject in need thereof, aneffective amount of at least one antagonist which modulates mGluR2,mGluR3, and mGluR5, or a combination according to claim 10, wherein theeffective amount is sufficient to diminish, inhibit or eliminate desirefor and/or consumption of the substance in the subject.
 21. The methodof claim 20, wherein the substance is nicotine, alcohol, opiates,amphetamines, methamphetamines, or cocaine.
 22. The method of claim 21,wherein LY341495 and 2-methyl-6-(phenylethynyl)-pyridine areadministered to the subject.
 23. A method of screening for an agent thatimproves the ability of a known inhibitor to at least partiallynormalize an intracranial self-stimulation (ICSS) threshold of anon-human mammalian subject, comprising: a) affecting the ICSS thresholdof the subject; b) administering to the subject, a sufficient amount ofthe known inhibitor to at least partially normalize the ICSS thresholdwhen administered alone or in combination with another inhibitor,wherein the known inhibitor is an antagonist of at least one of mGluR2,mGluR3, and mGluR5; c) administering to the non-human mammalian subject,an effective amount of a test agent, wherein the test agent is a knownor suspected antagonist of at least one of mGluR2,mGluR3, and mGluR5;and d) determining whether the test agent improves the ability of theknown inhibitor to at least partially normalize the ICSS threshold,thereby identifying an agent that improves the ability of the knowninhibitor to at least partially normalize ICSS threshold.
 24. The methodof claim 23, wherein the method identifies the test agent as an agenteffective for the treatment of depression or an addictive disorder. 25.The method of claim 23, wherein the known inhibitor is LY341495 or2-methyl-6-(phenylethynyl)-pyridine.
 26. The method of claim 23, whereinthe test agent improves the ability of the known inhibitor to inhibitdesire for and/or consumption of an addictive substance.
 27. A methodfor treating an addictive disorder, comprising: a) administering to asubject in need thereof, an effective amount of at least one antagonistthat modulates at least one of mGluR2, 3, and 5 during a first timeperiod, wherein the first time period is a time period wherein thesubject expects to be in an environment wherein, or exposed to stimuliin the presence of which, the subject habitually uses an addictivesubstance; and b) administering at least one antagonist that modulatesat least one of mGluR2 and/or 3 during a second time period, wherein thesecond time period is a time period wherein the subject is sufferingfrom withdrawal and/or depression.
 28. The method of claim 27, whereinone or both 2-methyl-6-(phenylethynyl)-pyridine and LY341495 areadministered during the first time period, and LY341495 is administeredduring the second time period.
 29. A method for treating depressivesymptoms and anxiety symptoms of depression, comprising administering toa subject in need thereof, an effective amount of at least oneantagonist which modulates metabotropic glutamate receptor 2,metabotropic glutamate receptor 3, and metabotropic glutamate receptor5, thereby treating the depressive symptoms and anxiety symptoms ofdepression.
 30. The method of claim 29, wherein an antagonist ofmetabotropic glutamate receptor 2 and metabotropic glutamate receptor 3is administered when the subject experiences depression symptoms, and anantagonist of metabotropic glutamate receptor 5 is administered when thesubject experiences anxiety symptoms.
 31. The method of claim 30,wherein LY341495 and 2-methyl-6-(phenylethynyl)-pyridine areadministered to the subject.